Columbia  (Mntoersrttp 
tntljpCitpuflfttigork 

College  of  ipfjpatctansi  anb  gmrgeona 


Htbrarp 


t resented by 

^DR.  WILLIAM  J.  GI 

to  enrich  the  library  resourcei 
available  to  holders 

of  the  Us 

GIES  FELLOWSHIP 

in  Biological  Chemistry 


PRACTICAL 
PHYSIOLOGICAL    CHEMISTRY 

HAWK 


Absorption  Spectra. 


PLATE    I. 


&     V 


£    /■■ 


Oxy  haemoglobin. 


Haemoglobin. 


Carboxy- 

haemoglobin. 


Neutral  Met- 

haemoglobin. 


Alkaline  Met- 

haemoglobin. 


Alkali 
Haeaatin. 


Absorption  Spectra. 


PLATE      II. 


n   '<■ 


/    / 


Reduced  Alkali 
Haematm  or 
HaemiK  hromoeen. 


Acid  Haematin  in 
ethereal  solution. 


Acid  Haemato- 
porphyrln. 


Alkaline 

Haematopor- 

phyrin. 


Urobilin  or  Hydro- 
bllirubin  in  acid 
solution. 


Urobilin  or  Hydro- 
bilirubin  in  alkaline 
solution  after  the 
addition  of  zinc 
chloride  solution. 


Bilicyanin  or 
Cholecyanin  in 
alkaline  solution. 


PRACTICAL 

PHYSIOLOGICAL    CHEMISTRY 


A  Book  UNSIGNED  FOR  USE  IN  COURSES 
IN  PRACTICAL  PHYSIOLOGICAL  CHEMISTRY 
IN  SCHOOLS  OF  MEDICINE  AND  OF  SCIENCE 


BY 

PHILIP    B.    HAWK.    M.S.,   Ph.D. 

DBMONSTRATOR    "l     PHYSIOLOGII  A  I.    I  HBMISTRV     IN    THB    DEPARTMENT   OF    Ml  I.I'  INI 
l  II  I     INI\  BR!  I  NSYLYANIA 


WITH   Tiro  FULL   PAGE  PLATES  OF  ABSORPTION  SPECTRA   IN  COLORS. 
:■:  ADDITIONAL  FULL  PACE  COLOR  PLATES  AND  ONE  HUN- 
DRED AND    TWENTY-SIX  FIGURES  OF  WHICH 
TWELVE  ARE  IN  COLORS 


PHILADELPHIA 

P.  BLAKISTON'S   SON    &   CO. 

IOI2    WALNUT    STREET 

1907 


Copyright  1907,  By  P.  Blakiston's  Son  &  Co. 


?s\4 


Press  of 

The  new  Era  Printing  Compam/ 

Lancaster,  Pa. 


THESE    PAGES 
ARE    AFFECTIONATELY    DEDICATED 

TO 

MY    MOTHER 


PREFACE 

The  plan  followed  in  the  presentation  of  the  subject  of  this 
volume  is  rather  different,  so  far  as  the  author  is  aware,  from 

that    set    forth,  in   any   similar    volume.      This   plan.    how< 
he  feels  to  be  a  logical  one  and  has  followed  it  with  satis 
tory  results  during  a  period  of  three  years  in  his  own  classes 
at  the  University  of  Pennsylvania.     The  main  point  in  which 
the  plan  of  the  author  differs  from  those  previously  pro], 
is  in  the  treatment  of  the  food  stuffs  and  their  digestion. 

In  Chapter  IV  the  "Decomposition  Products  of  Proteids" 
has  been  treated  although  it  is  impracticable  to  include  the 
study  of  this  topic  in  the  ordinary  course  in  practical  physio- 
logical chemistry.  For  the  specimens  of  the  decomposition 
products,  the  crystalline  forms  of  which  are  reproduced  by 
original  drawings  or  by  micro-photographs,  the  author  is  in- 
debted to  Dr.  Thomas  B.  Osborne,  of  Xew  Haven,  Conn. 

Because  of  the  increasing  importance  attached  to  the  ex- 
amination of  feces  for  purposes  of  diagnosis,  the  author  has 
devoted  a  chapter  to  this  subject.  He  feels  that  a  careful 
study  of  this  topic  deserves  to  he  included  in  the  courses  in 
practical  physiological  chemistry,  of  medical  schools  in  par- 
ticular. The  subject  of  solid  tissues  (Chapters  XIII.  XIV 
and  XV)  has  also  been  somewhat  more  fully  treated  than  has 
generally  been  customary  in  books  of  this  character. 

The  author  is  deeply  indebted  to  Professor  Lafayette  B. 
Mendel,  of  Yale  University,  for  his  careful  criticism  of  the 
manuscript  and  to  Professor  John  Marshall,  of  the  University 
of  Pennsylvania,  for  his  painstaking  revision  of  the  proof. 
He  also  wishes  to  express  his  gratitude  to  Dr.  David  L. 
Edsall  for  his  criticism  of  the  clinical  portion  of  the  volume ; 
to  Dr.  Otto  Folin  for  suggestions  regarding  several  of  his 
quantitative  methods,  and  to  Mr.  John  T.  Thomson  for  assist- 
ance in  proof-reading. 


Y11I  PHYSIOLOGICAL    CHEMISTRY. 

For  the  micro-photographs  of  oxyhemoglobin  and  haemm 
reproduced  in  Chapter  XI  the  author  is  indebted  to  Professor 
E.  T.  Reichert,  of  the  University  of  Pennsylvania,  who,  in 
collaboration  with  Professor  A.  P.  Brown,  of  the  University 
of  Pennsylvania,  is  making  a  very  extended  investigation  into 
the  crystalline  forms  of  biochemic  substances.  The  micro- 
photograph  of  allantoin  was  kindly  furnished  by  Professor 
Mendel.  The  author  is  also  indebted  for  suggestions  and 
assistance  received  from  the  lectures  and  published  writings 
of  numerous  authors  and  investigators. 

The  original  drawings  of  the  volume  were  made  by  Mr. 
Louis  Schmidt  whose  eminently  satisfactory  efforts  are  highly 
appreciated  by  the  author. 

Philip  B.  Hawk. 

Philadelphia,  March  27,  1907. 


CONTENTS. 


CHAPTER    I. 
(  Iarbohydrates I 

CHAPTER  II. 
Salivary    Digestion   32 

CHAPTER  III. 

1  *R(  ITEIDS     42 

CHAPTER  IV. 
Decomposition  Products  of  Proteids 65 

CHAPTER  V. 
Gastric  Digestion 83 

CHAPTER  VI. 
Fats   96 

CHAPTER  VII. 
Pancreatic  Digestion 106 

CHAPTER  VIII. 
Bile  116 

CHAPTER  IX. 
Putrefaction  Products  129 

CHAPTER  X. 
Feces   139 

CHAPTER  XI. 
Blood 148 

CHAPTER  XII. 
Milk   187 

ix 


X  CONTENTS. 

CHAPTER  XIII. 
Epithelial  and  Connective  Tissues 197 

CHAPTER  XIV. 
Muscular  Tissue 206 

CHAPTER  XV. 
Nervous  Tissue • 220 

CHAPTER  XVI. 
Urine:    General  Characteristics  of  Normal  and  Path- 
ological Urine 226 

CHAPTER  XVII. 
Urine  :    Physiological  Constituents 237 

CHAPTER  XVIII. 
Urine  :    Pathological  Constituents 282 

CHAPTER  XIX. 
Urine  :    Organized  and  Unorganized  Sediments 318 

CHAPTER  XX. 
Urine  :    Calculi 340 

CHAPTER  XXI. 
Urine  :    Quantitative  Analysis 344 

CHAPTER  XXII. 
Quantitative   Analysis   of    Milk,    Gastric   Juice   and 

Blood  38° 


LIST  OF  ILLUSTRATIONS. 

l'l     Ml. 

I     Absorption  Spectra.  ,         .     . 

Frontispiece 

1 1.  AbsorptK  m  bpectra. 

1 1 1 .  (  Isazons. .- Opp<  »site  page  5 

l\.   Normal  Erythrocytes  and  Leucocytes ....  Opposite  page  151 

V.  Uric  Acid  Crystals Opposite  page  247 

VI.  Ammonium  I  Irate Opposite  page  324 

Fig.  Pagi 

1.  Dialyzing  Apparatus  for  Students'  Use  6 

2.  Einhorn  Saccharometer  10 

3.  One  Form  of  Laurent  Polariscope 12 

4.  Diagrammatic    Representation    of    the    course    of    the 

Light  through  the  Laurent  Polariscope 13 

5.  Polariscope   |  Schmidt  and   I  lausch   Model )    14 

6.  Iodoform     21 

7.  PotaP >    Starch 23 

8.  I  '.can     Starch 23 

i).   Arrowroot    Starch 23 

10.  Rye    Starch 23 

1  1 .    I  larley    Starch 23 

12.  (  >at    Starch 23 

13.  Buckwheat    Starch 23 

14.  Mai/.e  Starch 23 

1  5.  Rice   Starch 27, 

16.  Pea    Starch 23 

17.  Wheat  Starch 23 

18.  Microscopical  Constituents  of  Saliva 36 

19.  Coagulation  Temperature  Apparatus 50 

20.  Edestin    54 

21.  Excelsin,  the  Proteid  of  the  Brazil  Nut 55 

22.  Fischer  Apparatus '  v 

23.  1  yrosin    

24.  Leucin    "9- 

25.  Aspartic    Acid 7° 


Xll  LIST    OF    ILLUSTRATIONS. 

26.  Glutamic  Acid 71 

27.  Glycocoll  Ester  Hydrochloride 72 

28.  Phenylalanin    y^ 

29.  L'sevo-a-Prolin 74 

30.  Copper  Salt  of  Prolin 75 

31-  Serin    75 

32.  Cystin    j6 

33.  Lysin    Picrate 78 

34.  Histidin   Hydrochloride 79 

35.  Beef  Fat 96 

36.  Mutton   Fat 99 

37.  Pork    Fat 101 

38.  Palmitic   Acid 102 

39.  Melting-Point   Apparatus 103 

40.  Bile   Salts 118 

41.  Bilirubin    (Hsematoidin) 119 

42.  Cholesterin    125 

43.  Taurin    126 

44.  Glycocoll    127 

45.  Ammonium   Chloride 133 

46.  Microscopical  Constituents  of  Feces 139 

47.  Hsematoidin  Crystals  from  Acholic  Stools 140 

48.  Charcot-Leyden   Crystals 141 

49.  Boas'    Sieve 143 

50.  Oxyhemoglobin    Crystals    from   Blood   of  the   Guinea 

Pig   152 

51.  Oxyhsemoglobin   Crystals  from   Blood  of  the  Rat....  152 

52.  Oxyhsemoglobin  Crystals  from  Blood  of  the  Horse.  ...  153 

53.  Oxyhsemoglobin  Crystals  from  Blood  of  the  Squirrel.  .  153 

54.  Oxyhsemoglobin  Crystals  from  Blood  of  the  Dog 154 

55.  Oxyhsemoglobin  Crystals  from  Blood  of  the  Cat 154 

56.  Oxyhsemoglobin  Crystals  from  Blood  of  the  Necturus.  .  155 

57.  Effect  of  Water  on  Erythrocytes 162 

58.  Hsemin  Crystals  from  Human  Blood 164 

59.  Hsemin  Crystals  from  Sheep  Blood 164 

60.  Sodium    Chloride    167 

61.  Direct-vision   Spectroscope 170 

62.  Angular-vision  Spectroscope  Arranged  for  Absorption 

Analysis    170 


LIST    OF    [LLUSTRATIONS.  xiii 

63.  Diagram  of  Angular-vision  Spectroscope 171 

64.  Fleischl's    Haemometer [75 

65.  Pipette  of  Fleischl's  1  [aem<  imeter 1 75 

66.  Colored  Glass  Wedge  of  Fleischl's  Haemometer [76 

6y.  Dare's    Haemoglobinometer 17s 

68.  Horizontal  Section  of  Dare's  Haemoglobinometer [79 

69.  Method  of    Filling  the  Capillary  Observation  Cell  of 

I  )are's    I  [aemoglobinometer 180 

70.  Tin  ima-Zeiss  Counting  Chamber t8i 

71.  Thoma-Zeiss  Capillary    Pipettes 1X2 

7_\  (  >rdinary  Ruling  of  Thoma-Zeiss  Counting  Chamber.  .  [83 

73.  Zappert's    Modified    Ruling  of    Thoma-Zeiss  Counting 

Chamber  [84 

74.  Normal  Milk  and  Colostrum 188 

j^.  Lactose   [89 

76.  Calcium    Phosphate. 193 

yy.  Crcatin    209 

78.  Xanthin   210 

79.  Hypoxanthin  Silver  Nitrate 216 

80..  Xanthin  Silver  Nitrate 218 

81.  Deposit  in  Ammoniacal  Fermentation 229 

82.  Deposit  in  Acid  Fermentation 22y> 

83.  Urinometer  and   Cylinder 231 

84.  Beckmann-Heiderihain  Freezing-Point  Apparatus 233 

85.  Urea    231 ) 

86.  Urea    Nitrate 242 

87.  Melting- Point  Tubes  Fastened  to  Bulb  of  Thermometer.  243 

88.  Urea    Oxalate 244 

89.  Pure  Uric  Acid 241  > 

90.  Creatinin    251 

iji .  Creatinin-Zinc  Chloride   252 

92.  Hippuric    Acid 

93.  Allantoin  from  Cat's  Urine 

94.  Benzoic    Acid 264 

95.  Calcium    Sulphate 274 

96.  "  Triple    Phosphate  " 278 

97.  The  Purdy  Electric  Centrifuge 318 

98.  Sediment  Tube  for  the  Purdy  Electric  Centrifuge 318 

99.  Calcium  Oxalate 32° 


XIV  LIST    OF    ILLUSTRATIONS. 

ioo.  Calcium    Carbonate 321 

101.  Various  Forms  of  Uric  Acid $23 

102.  Acid  Sodium  Urate 324 

103.  Cystin    325 

104.  Crystals  of  Impure  Leucin 326 

105.  Epithelium  from  Different  Areas  of  the  Urinary  Tract.  329 

106.  Pus   Corpuscles 330 

107.  Hyaline    Casts 331 

108.  Granular    Casts 332 

109.  Granular  Casts    333 

1 10.  Epithelial    Casts 333 

111.  Blood,  Pus.  Hyaline  and  Epithelial  Casts 334 

1 1 2.  Fatty    Casts 335 

113.  Fatty  and  Waxy  Casts 335 

1 14.  Cylindroids    336 

115.  Crenated   Erythrocytes 337 

1 16.  Human    Spermatozoa 338 

117.  Esbach's  Albuminometer  345 

118.  Marshall's  Urea  Apparatus 352 

1 19.  Hiifner's   Urea   Apparatus 354 

120.  Doremus-Hinds   Ureometer 355 

121.  Folin's  Urea  Apparatus 356 

122.  Folin's   Ammonia   Apparatus 358 

123.  Folin  Absorption  Tube 359 

124.  Berthelot-Atwater  Bomb  Calorimeter 366 

125.  Soxhlet  Apparatus 380 

126.  Feser's  Lactoscope 381 


PHYSIOLOGICAL  CHEMISTRY. 


CHAPTER    I. 

CARBOHYDRATES. 

Tiif:  name  carbohydrates  is  given  to  a  class  of  bodies  which 
are  an  especially  prominent  constituent  of  plants  and  which 
are  found  also  in  the  animal  body  either  free  or  as  an  integral 
part  of  various  proteids.  They  are  called  carbohydrates  be- 
cause they  contain  the  elements  C,  H  and  O ;  the  H  and  O 
being  present  in  the  proportion  to  form  water.  The  term 
is  not  strictly  appropriate  inasmuch  as  there  are  bodies  such 
as  acetic  acid,  lactic  acid  and  inosit  which  have  H  and  O 
present  in  the  proportion  to  form  water,  but  which  are  not 
carbohydrates,  and  there  are  also  true  carbohydrates  which  do 
not  have  H  and  O  present  in  this  proportion,  e.  g.,  rhamnose, 
C6H12Ob. 

Chemically  considered,  the  carbohydrates  are  aldehyde  or 
ketone  derivatives  of  complex  alcohols.  Treated  from  this 
standpoint  the  aldehyde  derivatives  are  spoken  of  as  aldoses, 
and  the  ketone  derivatives  are  spoken  of  as  ketoses.  The 
carbohydrates  are  also  frequently  named  according  to  the 
number  of  carbon  atoms  present  in  the  molecule,  e.  g.,  trioses, 
pentoses  and  hexoses. 

The  more  common  carbohydrates  may  be  classified  as 
follows : 

I.   Monosaccharides. 

i.  Hexoses,  C6H12Ofi. 

(a)  Dextrose. 

(b)  Lsevulose. 


2  PHYSIOLOGICAL    CHEMISTRY. 

(c)   Galactose. 
2.  Pentoses,  C5H10O5. 

(a)  Arabinose. 

(b)  Xylose. 

(c)  Rhamnose   (Methyl-pentose),  C6H1205. 
II.  Disaccharides,  C^H^On. 

i.  Maltose. 

2.  Saccharose. 

3.  Iso-Maltose. 

4.  Lactose. 

III.  Trisaccharides,  C18H32016. 

1.  Raffinose. 

IV.  Polysaccharides,  (C6H10O5)x. 

1.  Starch  Group. 

(a)  Starch. 

(b)  Inulin. 

(c)  Glycogen. 

(d)  Lichenin. 

2.  Gums  and  Vegetable  Mucilage  Group. 

(a)  Dextrin. 

(b)  Vegetable  Gums. 

3.  Cellulose  Group. 

(a)  Cellulose. 

(b)  Hemi-Cellulose. 

Each  member  of  the  above  carbohydrate  classes,  except 
the  members  of  the  pentose  group,  may  be  supposed  to  con- 
tain the  group  CcH10O5  called  the  saccharide  group.  The 
polysaccharides  consist  of  this  group  alone  taken  a  large  num- 
ber of  times,  whereas  the  disaccharides  may  be  supposed  to 
contain  two  such  groups  plus  a  molecule  of  water,  and  the 
monosaccharides  to  contain  one  such  group  plus  a  molecule 
of  water.  Thus,  (C0H10O5)x  —  polysaccharide,  (C6H10O5)2 
-j-  H20  =  disaccharide,  C6H10O5  +  H20  =  monosaccharide. 
In  a  general  way  the  solubility  of  the  carbohydrates  varies 
with  the  number  of  saccharide  groups  present,  the  substances 
containing  the  largest  number  of  these  groups  being  the  least 


MONOSACCHARIDES.  3 

soluble.  This  means  simply  that,  as  a  class,  the  monosac- 
charides (hexoses)  are  the  most  soluble  and  the  polysac- 
charides (starches  and  cellulose)  are  the  least  soluble. 


MONOSACCHARIDES. 

Hexoses,  C6Hi2Oe. 

The  hexoses  are  monosaccharides  containing-  six  carbon 
atoms  to  the  molecule.  They  are  the  most  important  of  the 
simple  sugars,  and  two  of  the  principal  hexoses.  dextrose  and 
lawulose,  occur  widely  distributed  in  plants  and  fruits.  These 
two  hexoses  also  result  from  the  hydrolysis  of  starch  and 
cane  sugar.  Galactose,  which  with  dextrose  results  from  the 
hydrolysis  of  lactose,  is  also  an  important  hexose.  These 
three  hexoses  are  fermentable  by  yeast,  and  yield  laevulinic 
acid  upon  heating  with  dilute  mineral  acids.  They  reduce 
metallic  oxides  in  alkaline  solution,  are  optically  active,  and 
extremely  soluble.  With  phenylhydrazin  they  form  charac- 
teristic osazons. 

CH2OH 
I 

DEXTROSE,   (CHOH)4. 
I 

CHO 

Dextrose,  also  called  glucose,  grape  sugar,  or  diabetic  sugar, 
is  present  in  the  blood  in  small  amount  and  may  also  occur 
in  traces  in  normal  urine.  After  the  ingestion  of  large 
amounts  of  saccharose,  lactose  or  dextrose  an  alimentary  gly- 
cosuria occasionally  arises.  In  diabetes  mellitus  very  large 
amounts  of  dextrose  are  excreted  in  the  urine.  The  fol- 
lowing structural  formula  has  been  suggested  by  Victor  Meyer 
for  (/-dextrose: 


4  PHYSIOLOGICAL    CHEMISTRY. 

COH 

I 
H  —  C  —  OH 

HO  —  C  —  H 

I 
H  —  C  —  OH 

H  —  C  —  OH 

I 
CH2OH 

(For  further  discussion  of  dextrose  see  section  on  Hexoses, 
page  3.) 

Experiments  on  Dextrose. 

1.  Solubility. — Test  the  solubility  of  dextrose  in  the  "  ordi- 
nary solvents "  and  in  alcohol.  (In  the  solubility  tests 
throughout  the  book  we  shall  designate  the  following  solvents 
as  the  "ordinary  solvents":  H20;  10  per  cent  NaCl;  0.5 
per  cent  Na2C03 ;  0.2  per  cent  HC1 ;  concentrated  KOH ; 
concentrated  HC1.) 

2.  Molisch's  Reaction. — Place  approximately  5  c.c.  of 
concentrated  H2S04  in  a  test-tube.  Incline  the  tube  and 
slowly  pour  down  the  inner  side  of  it  approximately  5  c.c. 
of  the  sugar  solution  to  which  2  drops  of  a-naphthol  solu- 
tion (about  15  per  cent  alcoholic  solution)  has  been  added, 
so  that  the  sugar  solution  will  not  mix  with  the  acid.  A 
reddish-violet  zone  is  produced  at  the  point  of  contact.  The 
reaction  is  due  to  the  formation  of  furfurol, 

HC  — CH 

II        II 
HC      C  -CHO, 
\  / 
0 

by  the  acid.  The  test  is  given  by  all  bodies  containing  a  car- 
bohydrate group  and  is  therefore  of  very  little  practical  im- 
portance. 


PLATE    III. 


OSAZONS. 

Upper   form,  dextrosazon  ;   central   form,  maltosazon  ;   lower   form,  lactosazon. 


MONOSACCHARIDES.  5 

3.  Phenylhydrazin  Reaction.— -Test  according  to  one  of 
the  following  methods:  (a)  To  a  small  amount  of  phenvl- 
hydrazin  mixture,  furnished  by  the  instructor,1  add  5  c.c.  of 
the  sugar  solution,  shake  well  and  heal  on  a  boiling  water- 
hath  for  "iK- halt"  to  three-quarters  of  an  hour.  Allow  the 
tube  to  cool  slowly  and  examine  the  crystals  microscopically 
(  Plate  111.  opposite).  If  the  solution  has  become  too  concen- 
trated in  the  boiling  process  it  will  be  light-red  in  color  and 
no  crystals  will  separate  until  it  is  diluted  with  water. 

Yellow  crystalline  bodies  called  osazons  are  formed  from 
certain  sugars  under  these  conditions,  each  individual  sugar 
giving  rise  to  an  osazon  of  a  definite  crystalline  form  which 
is  typical  for  that  sugar.  Each  osazon  has  a  definite  melting- 
point  and  as  a  further  and  more  accurate  means  of  identifica- 
tion it  may  be  recrystallized  and  identified  by  the  determination 
of  its  melting-point  and  nitrogen  content.  The  reaction  tak- 
ing place  in  the  formation  of  phciiyhlcxtrosazon  is  as  follows: 

C6H12Oc  +  2(H2N-NH-C6H5)  = 

Dextrose.  Phenylhydrazin. 

C0H10O4(N-NH-C6H5)2  +  2H20  +  H2. 

Phenyldextrosazon. 

(b)  Place  5  c.c.  of  the  sugar  solution  in  a  test-tube,  add  1  c.c. 
of  the  phenylhydrazin-acetate  solution  furnished  by  the  in- 
structor," and  heat  on  a  boiling  water-bath  for  one-half  to 
three-quarters  of  an  hour.  Allow  the  liquid  to  cool  slowly  and 
examine  the  crystals  microscopically  (Plate  III,  opposite). 

The  phenylhydrazin  test  has  been  so  modified  by  Cipollina 
as  to  be  of  use  as  a  rapid  clinical  test.  The  directions  for 
this  test  are  given  in  the  next  experiment. 

1This  mixture  is  prepared  by  combining  one  part  of  phenylhydrazin- 
hydrochloride  and  two  parts  of  sodium  acetate,  by  weight.  These  are 
thoroughly  mixed  in  a  mortar. 

2  This  solution  is  prepared  by  mixing  one  part  by  volume,  in  each  case, 
of  glacial  acetic  acid,  one  part  of  water  and  two  parts  of  phenylhydrazin 
(the  base). 


6  PHYSIOLOGICAL    CHEMISTRY. 

4.  Cipollina's  Test. — Thoroughly  mix  4  c.c.  of  dextrose 
solution,  5  drops  of  phenylhydrazin  (the  base)  and  ]/2  c.c. 
of  glacial  acetic  acid  in  a  test-tube.  Heat  the  mixture  for 
about  one  minute  over  a  low  flame,  shaking  the  tube  con- 
tinually to  prevent  loss  of  fluid  by  bumping.  Add  4-5  drops 
of  sodium  hydroxide  (sp.  gr.  1.16),  being  certain  that  the 
fluid  in  the  test-tube  remains  acid,  heat  the  mixture  again 
for  a  moment  and  then  cool  the  contents  of  the  tube.  Ordi- 
narily the  crystals  form  at  once,  especially  if  the  sugar  solu- 
tion possesses  a  low  specific  gravity.  If  they  do  not  appear 
immediately  allow  the  tube  to  stand  at  least  20  minutes  before 
deciding  upon  the  absence  of  sugar. 

Examine  the  crystals  under  the  microscope  and  compare 
them  with  those  shown  in  Plate  III,  opposite  page  5. 

5.  Precipitation  by  Alcohol. — To  10  c.c.  of  95  per  cent 
alcohol  add  about  2  c.c.  of  dextrose  solution.  Compare  the 
result  with  that  obtained  under  Dextrin,  7,  page  28. 

6.  Iodine  Test. — Make  the  regular  iodine  test  as  given 
under  Starch,  5,  page  24,  and  compare  this  result  with  the 
results  obtained  with  starch  and  with  dextrin. 

7.  Diffusibility  of  Dextrose. — Test  the  diffusibility  of  dex- 
trose solution  through  animal  membrane,  or  parchment  paper, 

Fig.  1. 


Dialyzixg  Apparatus  for  Students'  Use. 


making  a  dialyzer  like  one  of  the  models  shown  in  Fig.   1, 
above. 


MONOSACCHARIDES.  7 

8.  Moore's  Test. — To  2  3  c.c.  of  sugar  solution  in  a  ; 
tube  add  an  equal  volume  of  concentrated  K'MI  <>r  NaOH, 
and  boil.     The  solution  darkens  and  finally  assumes  a  brown 
color.     This  is  an  exceedingly  crude  tesl  and  is  of  little  prac- 
tical value. 

9.  Reduction  Tests. — To  their  aldehyde  or  ketone  struc 
tine  many  sugars  owe  the  property  of  readily  reducing  alka- 
line solutions  of  the  oxides  of  metals  like  copper, bismuth  and 
mercury ;  they  also  possess  the  property  of  reducing  ammo- 
niacal  silver  solutions  with  the  separation  of  metallic  silver. 
Upon  this  pmperty  of  reduction  the  most  widely  used  tests 
for  sugars  are  based.  When  whitish-blue  cupric  hydroxide 
in  suspension  in  an  alkaline  liquid  is  heated  it  is  converted 
into  insoluble  black  cupric  oxide,  but  if  a  reducing  agent  like 
certain  sugars  be  present  the  cupric  hydroxide  is  reduced  to 
insoluble  yellow  cuprous  hydroxide,  which  in  turn  on  further 
heating  may  be  converted  into  brownish-red  or  red  cuprous 
oxide.     These  changes  are  indicated  as  follows : 


OH 

/ 
Cu  m+  Cu=0  +  H20. 

\^  Cupric  oxide. 

OH  (black). 

Cupric  hydroxide, 
(whitish-blue). 


OH 

/ 
Cu 

\ 

OH 

=->-   2Cu-OH  +  H20  +  0. 

OH  Cuprous  hydroxide.. 

/  (yellow). 

Cu 

\ 

OH 


PHYSIOLOGICAL    CHEMISTRY. 


Cu-OH 
Cu-OH 

Cu 

\ 

0  +  H20 

/ 
Cu 

Cuprous  hydroxide 
(yellow). 

Cuprous  oxide, 
(brownish-red). 

The  chemical  equations  here  discussed  are  exemplified  in 
Trommer's  and  Fehling's  tests. 

(a)  Trommer's  Test. — To  5  c.c.  of  sugar  solution  in  a 
test-tube  add  one-half  its  volume  of  KOH  or  NaOH.  Mix 
thoroughly  and  add,  drop  by  drop,  a  very  dilute  solution  of 
cupric  sulphate.  Continue  the  addition  until  there  is  a  slight 
permanent  precipitate  of  cupric  hydroxide  and  in  consequence 
the  solution  is  slightly  turbid.  Heat,  and  the  cupric  hydroxide 
is  reduced  to  yellow  cuprous  hydroxide  or  to  brownish-red 
cuprous  oxide.  If  the  solution  of  cupric  sulphate  used  is  too 
strong  a  small  brownish-red  precipitate  produced  in  a  weak 
sugar  solution  may  be  entirely  masked.  On  the  other  hand, 
particularly  in  testing  for  sugar  in  the  urine,  if  too  little 
cupric  sulphate  is  used  a  light-colored  precipitate  formed  by 
uric  acid  and  purin  bases  may  obscure  the  brownish-red  pre- 
cipitate of  cuprous  oxide.  The  action  of  KOH  or  NaOH 
in  the  presence  of  an  excess  of  sugar  and  insufficient  copper 
will  produce  a  brownish  color.  Phosphates  of  the  alkaline 
earths  may  also  be  precipitated  in  the  alkaline  solution  and 
be  mistaken  for  cuprous  hydroxide.  Trommer's  test  is  not 
very  satisfactory. 

(b)  Fehling's  Test. — To  about  1  c.c.  of  Fehling's  solution1 

fehling's  solution  is  composed  of  two  definite  solutions — a  cupric 
sulphate  solution  and  an  alkaline  tartrate  solution,  which  may  be  prepared 
as  follows : 

Cupric  sulphate  solution  =  34.64  grams  of  cupric  sulphate  dissolved  in 
water  and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =  125  grams  of  potassium  hydroxide  and 
173  grams  of  Rochelle  salt  dissolved   in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bot- 
tles and  mixed  in  equal  volumes  when  needed  for  use.  This  is  done  to 
prevent  deterioration. 


MONOSACCHARIDES.  9 

in  a  test-tube  add  about  4  c.c.  of  water,  and  boil.     This  is 

done  to  determine  whether  the  solution  will  of  it-elf  cause  the 
formation  of  a  precipitate  of  brownish-red  cuprous  oxide.  If 
such  a  precipitate  forms,  the  Fehling's  solution  must  not  lie- 
used.  Add  sugar  solution  to  the  warm  Fehling's  solution  a 
few  drops  at  a  time  and  heat  the  mixture  after  each  addition. 
The  production  of  yellow  cuprous  hydroxide  or  brownish-red 
cuprous  oxide  indicates  that  reduction  has  taken  place.  The 
yellow  precipitate  is  more  likely  to  occur  if  the  sugar  solution 
is  added  rapidly  and  in  large  amount,  whereas  with  a  less 
rapid  addition  of  smaller  amounts  of  sugar  solution  the 
brownish-red  precipitate  is  generally  formed. 

This  is  a  much  more  satisfactory  test  than  Trommer's,  but 
even  this  test  is  not  entirely  reliable  when  used  to  detect  sugar 
in  the  urine.  Such  bodies  as  conjugate  glycuroiiates,  uric- 
acid,  nuclco-proteid  and  homogentisic  acid  when  present  in 
sufficient  amount  may  produce  a  result  similar  to  that  pro- 
duced by  sugar.  Phosphates  of  the  alkaline  earths  may  be 
precipitated  by  the  alkali  of  the  Fehling's  solution  and  in 
appearance  may  be  mistaken  for  cuprous  hydroxide.  Cupric 
hydroxide  may  also  be  reduced  to  cuprous  oxide  and  this  in 
turn  be  dissolved  by  crcatinin,  a  normal  urinary  constituent. 
This  will  give  the  urine  under  examination  a  greenish  tinge 
and  may  obscure  the  sugar  reaction  even  when  a  considerable 
amount  of  sugar  is  present. 

(c)  Boettger's  Test. — To  5  c.c.  of  sugar  solution  in  a  test- 
tube  add  1  c.c.  of  KOH  or  NaOH  and  a  very  small  amount 
of  bismuth  subnitrate,  and  boil.  The  solution  will  gradually 
darken  and  finally  assume  a  black  color  due  to  reduced  bis- 
muth. If  the  test  is  made  on  urine  containing  albumin  this 
must  be  removed,  by  boiling  and  filtering,  before  applying  the 
test,  since  with  albumin  a  similar  change  of  color  is  produced 
(bismuth  sulphide). 

(d)  Nylandcr's  Test  (Almens  Test). — To  5  c.c.  of  sugar 
solution  in  a  test-tube  add  one-tenth  its  volume  of  Nylander's 


IO 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  2. 


reagent1  and  boil  two  or  three  minutes.  The  solution  will 
darken  if  reducing  sugar  is  present  and  upon  standing  for  a 
few  moments  a  black  color  will  appear.  This  color  is  due  to 
the  precipitation  of  bismuth.  If  the  test  is  made  on  urine 
containing  albumin  this  must  be  removed,  by  boiling  and 
filtering,  before  applying  the  test.  It  is  claimed  by  Bechold 
that  Nylander's  and  Boettger's  tests  give  a  negative  reaction 
with  solutions  containing  sugar  when  mercuric  chloride  or 
chloroform  is  present,  a  claim  which  Zeidlitz  has  very  recently 
shown  to  be  incorrect. 

A  positive  Nylander  or  Boettger  test  is  probably  due  to  the 
following  reactions : 

(a)  Bi(OH)2N03  +  KOH  =  Bi(OH)3  +  KN03. 

(b)  2Bi(OH)3  —  30  =  Bi2  +  3H20. 

10.  Fermentation  Test.  —  "Rub 
up "  in  a  mortar  about  20  c.c.  of 
the  sugar  solution  with  a  small 
piece  of  compressed  yeast.  Transfer 
the  mixture  to  a  saccharometer 
(shown  in  Fig.  2)  and  stand  it  aside 
in  a  warm  place  for  about  twelve 
hours.  If  the  sugar  is  fermentable, 
alcoholic  fermentation  will  occur  and 
carbon  dioxide  will  collect  as  a  gas 
in  the  upper  portion  of  the  tube. 
On  the  completion  of  fermentation 
introduce  a  little  potassium  hydrox- 
ide solution  into  the  graduated  por- 
tion by  means  of  a  bent  pipette, 
place  the  thumb  tightly  over  the 
opening  in  the  apparatus  and  invert 
the  saccharometer.  Explain  the  re- 
sult. 

1  Nylander's  reagent  is  prepared  by  digesting  2  grams  of  bismuth  snb- 
nitrate  and  4  grams  of  Rochelle  salt  in  100  c.c.  of  a  10  per  cent  potas- 
sium hydroxide  solution.     The  reagent  is  then  cooled  and  filtered. 


Einiiorn    Saccharometer. 


MONOSACCHARIDES.  II 

ii.  Barfoed's  Test. — To  ->  3  cc.  of  Barfoed's  solution1  in 
a  test-tube  add  a  few  drops  of  dextrose  solution,  and  boil. 
Allow  to  stand  a  few  moments  and  examine.  Observe  tbe 
red  precipitate.     What  is  it? 

12.  Formation  of  Caramel. — Gently  heat  a  small  amount 
of  pulverized  dextrose  in  a  test-tube.  After  the  sugar  has 
melted  and  turned  brown,  allow  the  tube  to  cool,  add  water 
and  warm.  The  coloring  matter  produced  is  known  as 
caramel. 

13.  Demonstration  of  Optical  Activity. — A  demonstra- 
tion of  the  use  of  the  polariscope,  by  the  instructor,  each  stu- 
dent being  required  to  take  readings  and  compute  the  "  spe- 
cific rotation." 

Use  of  the  Polariscope. 

For  a  detailed  description  of  the  different  forms  of  polari- 
scopes,  the  method  of  manipulation  and  the  principles  in- 
volved the  student  is  referred  to  any  standard  text-book  of 
physics.  A  brief  description  follows :  An  ordinary  ray  of 
light  vibrates  in  every  direction.  If  such  a  ray  is  caused  to 
pass  through  a  "  polarizing  "  Nicol  prism  it  is  resolved  into 
tzvo  rays,  one  of  which  vibrates  in  every  direction  as  before 
and  a  second  ray  which  vibrates  in  one  plane  only.  This  latter 
ray  is  said  to  be  polarized.  Many  organic  substances  (sugars, 
proteids,  etc.)  have  the  power  of  twisting  or  rotating  this  plane 
of  polarized  light,  the  extent  to  which  the  plane  is  rotated 
depending  upon  the  number  of  molecules  which  the  polarized 
light  passes.  Substances  which  possess  this  power  are  said  to 
be  "  optically  active."  The  specific  rotation  of  a  substance 
is  the  rotation  expressed  in  degrees  which  is  afforded  by  one 
gram  of  substance  dissolved  in  1  cc.  of  water  in  a  tube  one 
decimeter  in  length.  The  specific  rotation,  (a)„,  may  be 
calculated  by  means  of  the  following  formula, 

1  Barfoed's  solution  is  prepared  as  follows:  Dissolve  4  grams  of  copper 
acetate  in  100  cc.  of  water  and  acidify  with  acetic  acid. 


12 


PHYSIOLOGICAL    CHEMISTRY. 


(«)»- 


^•/' 


in  which 

-o  =  sodium  light. 

a  =  observed  rotation  in  degrees. 

p  =  grams  of  substance  dissolved  in  I  c.c.  of  liquid. 

/  =  length  of  the  tube  in  decimeters. 
If  the  specific  rotation  has  been  determined  and  it  is  desired 
to  ascertain  the  per  cent  of  the  substance  in  solution,  this 
may  be  obtained  by  the  use  of  the  following  formula, 


The  value  of  p  multiplied  by  100  will  be  the  percentage  of  the 
substance  in  solution. 

An  instrument  by  means  of  which  the  extent  of  the  rota- 
tion may  be  determined  is  called  a  polariscope  or  polarimeter. 
Such  an  instrument  designed  especially  for  the  examination 
of  sugar  solutions  is  termed  a  saccharimeter  or  polarising  sac- 
cliarimeter.     The  form  of  polariscope  shown  in  Fig.  3,  below, 

Fig.  3. 


One  Form  of  Laurent  Polariscope. 
B,    Microscope    for   reading   the    scale ;    C,   a   vernier ;    E,   position    of   the 
analyzing    Nicol    prism ;    H,    polarizing    Nicol    prism    in    the    tube    below    this 
point. 


MONOSACCIIAUIIH.S.  13 

consists  essentially  of  a  long  barrel  provided  with  a  Nicol 
prism  at  either  end    (Fig.  4.  below).     The  solution   under 

examination  is  contained  in  a  tube  which  is  placed  between 
these  two  prisms.  At  the  front  end  of  the  instrument  is  an 
adjusting"  eye-piece  for  focusing  and  a  large  recording  disc 
which  registers  in  degrees  and  fractions  of  a  degree.  The 
light  is  admitted  into  the  far  end  of  the  instrument  and  is 
polarized  by  passing-  through  a  Nicol  prism.  This  polarized 
ray  then  traverses  the  column  of  liquid  within  the  tube  men- 
tioned above  and  if  the  substance  is  optically  active  the  plane 
of  the  polarized  ray  is  rotated  to  the  right  or  left.  Bodies 
rotating  the  ray  to  the  right  are  called  dextro-rotatory  and 
those  rotating  it  to  the  left  lecvo-rotatory. 


Fig.  4. 


(D 


/  1 


Diagrammatic  Representation  of  the  Course  of  the  Light  through  the 
Laurent  Polariscope.     (The  direction  is  reversed  from  that  of  Fig.  3,  p.  12.) 
a,   Bichromate  plate  to  purify  the  light ;   b,  the  polarizing  Nicol  prism  ;   c, 

a    thin   quartz    plate    covering    one-half    the    field    and    essential    in    producing 

a  second   polarized  plane ;   d,  tube  to   contain   the   liquid   under   examination ; 

e,  the  analyzing  Nicol  prism  ;  f  and  g,  ocular  lenses. 

Within  the  apparatus  is  a  disc  which  is  so  arranged  as  to 
be  without  lines  and  uniformly  light  at  zero.  Upon  placing 
the  optically  active  substance  in  position,  however,  the  plane 
of  polarized  light  is  rotated  or  turned  and  it  is  necessary 
to  rotate  the  disc  through  a  certain  number  of  degrees  in 
order  to  secure  the  normal  conditions,  i.  c  "  without  lines  and 
uniformly  light."  The  difference  between  this  reading  and 
the  zero  is  a  or  the  observed  rotation  in  degrees. 

Polarizing  saccharimeters  are  also  constructed  by  which  the 
percentage  of  sugar  in  solution  is  determined  by  making  an 
observation  and  multiplying  the  value  of  each  division  on  a 


H 


PHYSIOLOGICAL    CHEMISTRY. 


horizontal  sliding-  scale  by  the  value  of  the  division  expressed 
in  terms  of  dextrose.  The  value,  in  terms  of  dextrose,  of  each 
of  the  divisions  on  the  scale  of  the  Laurent  saccharimeter  used 
in  the  laboratory  of  physiological  chemistry  at  the  University 


Fig.  s 


Polariscope  (Schmidt  and  Hansch  Model). 

of  Pennsylvania  is  0.2051.     This  factor  may  vary  according 
to  the  instrument.  qjj  q-jj 

I 
LJEVULOSE,  (CHOH)3. 

I 

CO 

I 

CH2OH 

As  already  stated,  lsevulose,  sometimes  called  fructose  or 
fruit  sugar,  occurs  widely  disseminated  throughout  the  plant 


MONOSACCHARIDES.  1 5 

kingdom  in  company  with  dextrose.  Its  reducing-  power  is 
somewhat  weaker  than  that  of  dextrose.  Lawulose  does  not 
ordinarily  occur  in  the  urine  in  diabetes  mellitus-,  lmt  has  been 
found  in  exceptional  cases.  With  phenylhydrazin  it  forms  the 
same  osazon  as  dextrose.  With  methylphenylhydrazin,  laevu- 
lose  forms  a  characteristic  lsevulose-methylphenylosazon. 

(For  a  further  discussion  of  kevulose  see  the  section  on 
Hexoses,  p.  3.) 

Experiments  on  L.evulose. 

1.  Seliwanoff's  Reaction. — If  a  solution  of  resorcin  in 
dilute  HC1  (1  vol.  concentrated  1 1  CI  to  2  vols.  H20),  be 
warmed  with  lsevulose  the  liquid  will  become  red  and  a  pre- 
cipitate will  separate.  The  precipitate  may  be  dissolved  in 
alcohol  to  which  it  will  impart  a  striking  red  color. 

2.  Phenylhydrazin  Test. — Make  the  test  according  to  di- 
rections under  Dextrose,  3  or  4,  pages  5  and  6. 

CH2OH 

I 

GALACTOSE,  (CHOH)4. 

CHO 

Galactose  occurs  with  dextrose  as  one  of  the  products  of  the 
hydrolysis  of  lactose.  It  is  dextro-rotatory,  forms  an  osazon 
with  phenylhydrazin  and  ferments  slowly  with  yeast. 

Experiments  on  Galactose. 

1.  Tollens'  Reaction. — To  5  c.c.  of  hydrochloric  acid. 
having  a  specific  gravity  of  1.09,  add  a  slight  excess  of 
phloroglucin,  the  acid  being  kept  on  a  boiling  water-bath 
during  the  addition.  A  few  cubic  centimeters  of  galactose 
solution  should  now  be  added  and  the  heating  continued.  A 
red  color  is  produced.  Compare  this  color  with  that  given 
by  pentoses  (see  page  16). 

2.  Phenylhydrazin  Test. — Make  the  test  according  to  di- 
rections given  under  Dextrose,  3  or  4,  pages  5  and  6. 


1 6  PHYSIOLOGICAL    CHEMISTRY. 

Pentoses,  C5H10O5. 

In  plants  and  more  particularly  in  certain  gums,  very  com- 
plex carbohydrates,  called  pentosans,  occur.  These  pentosans 
through  hydrolysis  by  acids  may  be  transformed  into  pentoses. 
Pentoses  do  not  ordinarily  occur  in  the  animal  organism,  but 
have  been  found  in  the  urine  of  morphine  habitues  and  others, 
their  occurrence  sometimes  being  a  persistent  condition  with- 
out known  cause.  They  are  non-fermentable,  have  strong 
reducing  power,  and  form  osazons  with  phenylhydrazin. 
Pentoses  are  an  important  constituent  of  the  dietary  of  her- 
bivorous animals.  Glycogen  is  said  to  be  formed  after  the 
ingestion  of  these  sugars  containing  five  carbon  atoms.  On 
distillation  with  strong  hydrochloric  acid  pentoses  and  pen- 
tosans yield  furfurol,  which  can  be  detected  by  its  character- 
istic red  reaction  with  aniline-acetate  paper. 

CH,OH 

I 

ARABINOSE,   (CHOH)3. 
I 

CHO 

Arabinose,  one  of  the  most  important  pentoses,  may  be  ob- 
tained from  gum  arabic,  plum  or  cherry  gum  by  boiling  for 
several  hours  with  1-2  per  cent  sulphuric  acid.  It  is  dextro- 
rotatory, forms  an  osazon  and  has  reducing  power. 

Experiments  on  Arabinose. 

1.  Tollens'  Reaction. — To  equal  volumes  of  arabinose 
solution  and  hydrochloric  acid  (sp.  gr.  1.09)  add  a  little  phlor- 
oglucin  and  heat  the  mixture  on  a  boiling  water-bath.  Galac- 
tose, laevulose,  pentose  or  glycuronic  acid  will  be  indicated  by 
the  appearance  of  a  red  color.  To  differentiate  between  these 
bodies  make  a  spectroscopic  examination  and  look  for  the 
absorption  band  between  D  and  E  given  by  pentoses  and  gly- 
curonic acid.  Differentiate  between  the  two  latter  bodies  by 
the  melting-points  of  their  osazons. 


DISACCIIARIDES.  1 7 

Compare  the  reaction  with  that  obtained  with  galactose 
i  page  15 ). 

2.  Orcin  Test. — Repeat  i,  using  orcin  instead  of  pliloro- 
glucin.  A  succession  of  colors  from  red  through  reddish- 
blue  to  green  is  produced.  A  green  precipitate  is  formed 
which  is  soluble  in  amyl  alcohol  and  has  absorption  hand- 
between  C  and  D. 

3.  Phenylhydrazin  Test. — Make  this  test  on  the  arabinose 
solution  according  to  directions  given  under  Dextrose,  3  or  4. 
pages  g  and  6. 

CHoOH 
I 

XYLOSE,    (CHOH)3. 

I 

CHO 

Xylose,  or  wood  sugar,  is  obtained  by  boiling  wood  gums 
with  dilute  acids  as  explained  under  Arabinose,  page  16.  It 
is  dextro-rotatory  and  forms  an  osazon. 

Experiments  on  Xylose. 
1-3.  Same  as  for  arabinose  (see  page  16). 

RHAMNOSE,   C(,H]205. 

Rhamnose  or  methyl-pentose  is  an  example  of  a  true  carbo- 
hydrate which  does  not  have  the  H  and  O  atoms  present  in  the 
proportion  to  form  water.  Its  formula  is  CcH1205.  It  has 
been  found  that  rhamnose  when  ingested  by  rabbits  or  hens 
has  a  positive  influence  upon  the  formation  of  glycogen  in 
those  organisms. 

DISACCHARIDES,  C12H22On. 

The  disaccharides  as  a  class  may  be  divided  into  two  rather 
distinct  groups.     The  first  group  would  include  those  disac- 
charides which  are  found  in  nature  as  such.  e.  £.,  saccharose 
and  lactose,  and  the  second  group  would  include  those  disac- 
3 


l8  PHYSIOLOGICAL    CHEMISTRY. 

charides  formed  in  the  hydrolysis  of  more  complex  carbohy- 
drates, c.  g.j  maltose  and  iso-maltose. 

The  disaccharides  have  the  general  formula  C12H22011,  to 
which,  in  the  process  of  hydrolysis,  a  molecule  of  water  is 
added  causing  the  single  disaccharide  molecule  to  split  into 
two  monosaccharide  (hexose)  molecules. 

All  of  the  more  common  disaccharides  except  saccharose 
have  the  power  of  reducing  certain  metallic  oxides  in  alkaline 
solution,  notably  those  of  copper  and  bismuth.  This  reducing 
power  is  due  to  the  presence  of  the  aldehyde  group  ( —  CHO) 
in  the  sugar  molecule. 

MALTOSE,    CisHssOu. 

Maltose  or  malt  sugar  is  formed  in  the  hydrolysis  of 
starch  through  the  action  of  a  ferment,  diastase,  contained  in 
sprouting  barley  or  malt.  Certain  enzymes  in  the  saliva  and 
in  the  pancreatic  juice  may  also  cause  a  similar  hydrolysis. 
Maltose  is  also  an  intermediate  product  of  the  action  of  dilute 
mineral  acids  upon  starch.  It  is  strongly  dextro-rotatory,  re- 
duces metallic  oxides  in  alkaline  solution  and  is  fermentable 
by  yeast  after  being  inverted  (see  Polysaccharides,  page  21) 
by  the  enzyme  maltase  of  the  yeast.  In  common  with  the 
other  disaccharides,  maltose  may  be  hydrolyzed  with  the  for- 
mation of  two  molecules  of  monosaccharide.  In  this  instance 
the  products  are  two  molecules  of  dextrose.  With  phenylhy- 
drazin  maltose  forms  an  osazon,  maltosazon. 

Experiments  on  Maltose. 
1— II.   Repeat  these  experiments  as  given  under  Dextrose, 

pages  4-1 1. 

iso-maltose,  012H22011. 

Iso-maltose,  an  isomeric  form  of  maltose,  is  formed,  along 
with  maltose,  by  the  action  of  diastase  upon  starch  paste,  and 
also  by  the  action  of  hydrochloric  acid  upon  dextrose.  It  also 
occurs  with  maltose  as  one  of  the  products  of  salivary  diges- 
tion.    It  is  dextro-rotatory  and  with  phenylhydrazin  gives  an 


DISACCIIARIDES.  1 9 

osazon  which  is  characteristic,  [so-maltose  is  very  soluble 
and  reduces  the  oxides  of  bismuth  and  copper  ill  alkaline  solu- 
tion.    Pure  iso-maltose  is  probably  only  slightly  fermentable. 

LACTOSE,    CjjgHajOx!. 

Lactose  or  milk  sugar  occurs  ordinarily  only  in  milk,  but 
has  often  been  found  in  the  urine  of  women  during  pregnancy 
and  lactation.  It  may  also  occur  in  the  urine  of  normal  per- 
sons after  the  ingestion  of  unusually  large  amounts  of  lactose 
in  the  food.  It  has  a  strong  reducing  power,  is  dextro- 
rotatory and  forms  an  osazon  with  phenylhydrazin.  Upon 
hydrolysis  lactose  yields  one  molecule  of  dextrose  and  one 
molecule  of  galactose. 

In  the  souring  of  milk  the  bacterium  lactis  and  certain  other 
micro-organisms  bring  about  lactic  acid  fermentation  by 
transforming  the  lactose  of  the  milk  into  lactic  acid, 

H     OH 

I        I 
H  — C  — C  — COOH, 

I       I 
H     H 

and  alcohol.  This  same  reaction  may  occur  in  the  alimentary 
canal  as  the  result  of  the  action  of  putrefactive  bacteria.  In 
the  preparation  of  kephyr  and  koumyss  the  lactose  of  the  milk 
undergoes  alcoholic  fermentation,  through  the  action  of  fer- 
ments other  than  yeast,  and  at  the  same  time  lactic  acid  is 
produced. 

Lactose  is  not  fermentable  by  pure  yeast. 

Experiments  on  Lactose. 
I— II.     Repeat  these  experiments  as  given  under  Dextrose, 
pages  4-1 1. 

SACCHAROSE,   C^EUoOn. 

Saccharose,  also  called  sucrose  or  cane  sugar,  is  one  of  the 
most  important  of  the  sugars  and  occurs  very  extensively 


20  PHYSIOLOGICAL    CHEMISTRY. 

distributed  in  plants,  particularly  in  the  sugar  cane,  sugar 
beet,  sugar  millet  and  in  certain  palms  and  maples. 

Saccharose  is  dextro-rotatory  and  upon  hydrolysis,  as  be- 
fore mentioned,  the  molecule  of  saccharose  takes  on  a  mole- 
cule of  water  and  breaks  down  into  two  molecules  of  mono- 
saccharide. The  monosaccharides  formed  in  this  instance  are 
dextrose  and  laevulose.    This  is  the  reaction : 

C12H22On  +  H20  =  CnH1206  +  C6H12Ofi. 

Saccharose.  Dextrose.  Laevulose. 

This  process  is  called  inversion  and  may  be  produced  by  weak 
acids,  ferments  and  bacteria.  After  this  inversion  the  pre- 
viously strongly  dextro-rotatory  solution  may  be  lsevo- 
rotatory. 

Saccharose  does  not  reduce  metallic  oxides  in  alkaline  solu- 
tion and  forms  no  osazon  with  phenylhydrazin.  It  is  not  fer- 
mentable directly  by  yeast,  but  must  first  be  inverted  by  the 
ferment  invertin  contained  in  the  yeast. 

Experiments  on  Saccharose. 

I— II.  Repeat  these  experiments  according  to  the  directions 
given  under  Dextrose,  pages  4-1 1. 

12.  Inversion  of  Saccharose. — To  25  c.c.  of  saccharose 
solution  in  a  beaker  add  5  drops  of  concentrated  HC1  and  boil 
one  minute.  Cool  the  solution,,  render  alkaline  with  solid 
KOH  and  upon  the  resulting  fluid  repeat  experiments  3  (or 
4)  and  9  as  given  under  Dextrose,  pages  5,  6  and  7.  Explain 
the  results. 

13.  Production  of  Alcohol  by  Fermentation. — Prepare  a 
strong  (10-20  per  cent)  solution  of  saccharose,  add  a  small 
amount  of  egg  albumin  or  commercial  peptone  and  introduce 
the  mixture  into  a  bottle  of  appropriate  size.  Add  yeast,  and 
by  means  of  a  bent  tube  inserted  through  a  stopper  into  the 
neck  of  the  bottle,  conduct  the  escaping  gas  into  water.  As 
fermentation  proceeds  readily  in  a  warm  place  the  escaping 


POLYSACCHARIDES. 


21 


Fic.  6. 


gas   may  be   collected    in   a   eudiometer   tube   and    examined. 

When  the  activity  of  the  yeast    lias  practically  ceased,   filter 

the  contents  of  the  bottle   into   a 

flask  and  distil  the  mixture.     Catch 

the   first    portion    of    the    distillate 

separately  and  test  for  alcohol  by 

one  of  the  following  reactions: 

(a)  Iodoform  Test.  —  Render 
J  v}  c.c.  of  the  distillate  alkaline 
with  potassium  hydroxide  solution 
and  add  a  few  drops  of  iodine  so- 
lution. Heat  gently  and  note  the 
formation  of  iodoform  crystals. 
Examine  these  crystals  under  the 

microscope  and  compare  them  with  those  in  Fig.  6. 

(b)  Aldehyde  Test. — Place  5  c.c.  of  the  distillate  in  a  test- 
tube,  add  a  few  drops  of  potassium  dichromate  solution, 
K2Cr207,  and  render  it  acid  with  dilute  sulphuric  acid.  Boil 
the  acid  solution  and  note  the  odor  of  aldehyde. 


Iodoform.     (Autenrieth.  1 


TRISACCHARIDES,  C18H320lc. 

RAFFINOSE. 

This  trisaccharide,  also  called  melitose  or  melitriose,  occurs 
in  cotton  seed,  Australian  manna  and  in  the  molasses  from 
the  preparation  of  beet  sugar.  It  is  dextro-rotatory,  does  not 
reduce  Fehling's  solution  and  is  only  partially  fermentable 
by  yeast. 

Raffinose  may  be  hydrolyzed  by  weak  acids  the  same  as  the 
polysaccharides  are  hydrolyzed,  the  products  being  dextrose 
and  melibiose;  further  hydrolysis  of  the  melibiose  yields  dex- 
trose and  galactose. 


POLYSACCHARIDES,    (C6H1005)x. 

In  general  the  polysaccharides  are  amorphous  bodies,  a  few, 
however,  are  crystallizable.     Through  the  action  of  certain 


2  2  PHYSIOLOGICAL    CHEMISTRY. 

enzymes  or  weak  acids  the  polysaccharides  may  be  hydrolyzed 
with  the  formation  of  monosaccharides.  As  a  class  the  poly- 
saccharides are  quite  insoluble  and  are  non-fermentable  until 
inverted.  By  inversion  is  meant  the  hydrolysis  of  disaccharide 
or  polysaccharide  sugars  to  form  monosaccharides,  as  indi- 
cated in  the  following  equations  : 

(a)  C12H22On+H20  =  2(C6H1206)- 

(b)  C6H10O5  +  H2O  =  C6H12OG. 

STARCH,     (CGH10O5)x. 

Starch  is  widely  distributed  throughout  the  vegetable  king- 
dom, occurring  in  grains,  fruits  and  tubers.  It  occurs  in 
granular  form,  the  microscopical  appearance  being  typical  for 
each  individual  starch.  The  granules,  which  differ  in  size 
according  to  the  source,  are  composed  of  alternating  concen- 
tric rings  of  granulose  and  cellulose.  Ordinary  starch  is  in- 
soluble in  cold  water,  but  if  boiled  with  water  the  cell  walls  are 
ruptured  and  starch  paste  results. 

Starch  is  acted  upon  by  diastatic  enzymes,  e.  g.,  ptyalin  and 
a  my  I  opsin,  with  the  formation  of  soluble  starch,  erythro- 
dextrin,  achroo-dextrin,  malto-dextrin,  maltose,  iso-maltose 
and  dextrose  (see  Salivary  Digestion,  page  34).  Maltose  is 
the  principal  end-product  of  this  enzyme  action.  Upon  boil- 
ing a  starch  solution  with  a  dilute  mineral  acid  a  series  of 
similar  bodies  is  formed,  but  under  these  conditions  dextrose 
is  the  principal  end-product. 

Experiments  on  Starch. 

1.  Preparation  of  Potato  Starch. — Pare  a  raw  potato, 
comminute  it  upon  a  fine  grater,  mix  with  water,  and  "  whip 
up"  the  pulped  material  vigorously  before  straining  it  through 
cheese  cloth  or  gauze  to  remove  the  coarse  particles.  The 
starch  rapidly  settles  to  the  bottom  and  can  be  washed  by  re- 
peated decantation.  Allow  the  compact  mass  of  starch  to 
drain  thoroughly  and  spread  it  out  on  a  watch  glass  to  dry  in 


Pea.  Wheat. 

Starch  Granules  from  Various  Sources.     (LefFmann  and  Ream.) 


24  PHYSIOLOGICAL    CHEMISTRY. 

the  air.     If  so  desired  this  preparation  may  be  used  in  the  ex- 
periments which  follow. 

2.  Microscopical  Examination. — Examine  microscopic- 
ally the  granules  of  the  various  starches  submitted  and  compare 
them  with  those  shown  in  Figs.  7-17,  page  23. 

3.  Solubility. — Try  the  solubility  of  one  form  of  starch  in 
each  of  the  ordinary  solvents  (see  page  4).  If  uncertain 
regarding  the  solubility  in  any  reagent,  filter  and  test  the  fil- 
trate with  iodine  solution  as  given  under  5  below.  The  pro- 
duction of  a  blue  color  would  indicate  that  the  starch  had  been 
dissolved  by  the  solvent. 

4.  Iodine  Test. — Place  a  few  granules  of  starch  in  one  of 
the  depressions  of  a  porcelain  test-tablet  and  treat  with  a  drop 
of  a  dilute  solution  of  iodine  in  potassium  iodide.  The  gran- 
ules are  colored  blue  due  to  the  formation  of  so-called  iodide 
of  starch.  The  cellulose  of  the  granule  is  not  stained  as  may 
be  seen  by  examining  microscopically. 

5.  Iodine  Test  on  Starch  Paste. — Repeat  the  iodine  test 
using  the  starch  paste.  Place  2-3  c.c.  of  starch  paste1  in  a 
test-tube,  add  a  drop  of  the  dilute  iodine  solution  and  observe 
the  production  of  a  blue  color.  Heat  the  tube  and  note  the 
disappearance  of  the  color.     It  reappears  on  cooling. 

In  similar  tests  note  the  influence  of  alcohol  and  of  alkali 
upon  the  so-called  iodide  of  starch. 

The  composition  of  the  iodide  of  starch  is  not  definitely 
known. 

6.  Fehling's  Test. — On  starch  paste  (see  page  8). 

7.  Hydrolysis  of  Starch. — Place  about  25  c.c.  of  starch 
paste  in  a  small  beaker,  add  10  drops  of  concentrated  HC1,  and 
boil.  By  means  of  a  small  pipette,  at  the  end  of  each  minute, 
remove  a  drop  of  the  solution  to  the  test-tablet  and  make 

1  Preparation  of  Starch  Paste. — Grind  2  grams  of  starch  powder  in  a 
mortar  with  a  small  amount  of  cold  water.  Bring  200  c.c.  of  water  to  the 
boiling-point  and  add  the  starch  mixture  from  the  mortar  with  continuous 
stirring.  Bring  again  to  the  boiling-point  and  allow  it  to  cool.  This  makes 
an  approximate  1  per  cent  starch  paste  which  is  a  very  satisfactory 
strength  for  general  use. 


POLYSACCHARIDES.  25 

the  regular  iodine  test.  As  the  testing  proceeds  the  blue 
color  should  gradually  fade  and  finally  disappear.  At  this 
point,  after  cooling  and  neutralizing  with  solid  KOH,  Fehl- 
ing's  test  (see  p.  8)  should  give  a  positive  result  due  to  the 
formation  of  a  reducing  sugar  from  the  starch.  Make  the 
phenylhydrazin  test  upon  some  of  the  hydrolyzed  starch.  Try 
also  Barfoed's  test  (see  p.  11).     What  sugar  has  been  formed? 

8.  Influence  of  Tannic  Acid.— Add  an  excess  of  tannic 
acid  solution  to  a  small  amount  of  starch  paste  in  a  test-tube. 
The  liquid  will  become  strongly  opaque  and  ordinarily  a  yel- 
lowish-white precipitate  is  produced.  Compare  this  result 
with  the  result  of  the  similar  experiment  on  dextrin  (p.  28). 

9.  Diffusibility  of  Starch  Paste. — Test  the  diffusibility  of 
starch  paste  through  animal  membrane  or  parchment  paper, 
making  a  dialyzer  like  one  of  the  models  shown  in  Fig.  1, 
page  6. 

INULIN,    (C6H10OB)x 

Inulin  is  a  polysaccharide  which  may  be  obtained  as  a 
white,  odorless,  tasteless  powder  from  the  tubers  of  the  arti- 
choke, elecampane  or  dahlia.  It  has  also  been  prepared  from 
the  roots  of  chicory,  dandelion  and  burdock.  It  is  very  slightly 
soluble  in  cold  water  and  quite  easily  soluble  in  hot  water.  In 
cold  alcohol  of  60  per  cent  or  over  it  is  practically  insoluble. 
Inulin  gives  a  negative  reaction  with  iodine  solution.  The 
"yellow"  color  reaction  with  iodine  mentioned  in  many  books 
is  doubtless  merely  the  normal  color  of  the  iodine  solution. 
It  is  very  difficult  to  prepare  inulin  which  does  not  reduce 
Fehling's  solution  slightly.  This  reducing  power  may  be  due 
to  an  impurity.  Practically  all  commercial  preparations  of 
inulin  possess  considerable  reducing  power. 

Inulin  is  laevo-rotatory  and  upon  hydrolysis  by  acids  or  by 
the  enzyme  inulase  it  yields  the  monosaccharide  laevulose 
which  readily  reduces  Fehling's  solution.  The  ordinary 
amylolytic  enzymes  occurring  in  the  animal  body  do  not  digest 
inulin. 


26  physiological  chemistry. 

Experiments  on  Inulin. 
i.  Solubility. — Try  the  solubility  of  inulin  powder  in  each 
of  the  ordinary  solvents.  If  uncertain  regarding-  the  solu- 
bility in  any  reagent,  filter  and  neutralize  the  filtrate  if  it  is 
alkaline  in  reaction.  Add  a  drop  of  concentrated  hydrochloric 
acid  to  the  filtrate  and  boil  it  for  one  minute.  Render  the 
solution  neutral  or  slightly  alkaline  with  solid  KOH  and  try 
Fehling's  test.  AYhat  is  the  significance  of  a  positive  Fehling's 
test  in  this  connection? 

2.  Iodine  Test. —  (a)  Place  2-3  c.c.  of  the  inulin  solution 
in  a  test-tube  and  add  a  drop  of  dilute  iodine  solution.  What 
do  you  observe? 

(b)  Place  a  small  amount  of  inulin  powder  in  one  of  the 
depressions  of  a  test-tablet  and  add  a  drop  of  dilute  iodine 
solution.    Is  the  effect  any  different  from  that  observed  above? 

3.  Molisch's  Reaction. — Repeat  this  test  according  to  di- 
rections given  under  Dextrose.  2,  page  4. 

4.  Fehling's  Test. — Make  this  test  on  the  inulin  solution 
according  to  the  instructions  given  under  Dextrose,  page  8. 
Is  there  any  reduction  ? 1 

5.  Hydrolysis  of  Inulin. — Place  5  c.c.  of  inulin  solution 
in  a  test-tube,  add  a  drop  of  concentrated  hydrochloric  acid 
and  boil  it  for  one  minute.  Xow  cool  the  solution,  neutralize 
it  with  concentrated  KOH  and  test  the  reducing  action  of  1 
c.c.  of  the  solution  upon  1  c.c.  of  diluted  (1:4)  Fehling's 
solution.    Explain  the  result.2 

GLYCOGEN,    (C0H10O5)x. 
(For    discussion    and    experiments    see    Muscular    Tissue, 
page  206.) 

3  See  the  discussion  of  the  properties  of  inulin.  page  25. 

2  If  the  inulin  solution  gave  a  positive  Fehling  test  in  the  last  experi- 
ment it  will  be  necessary  to  check  the  hydrolysis  experiment  as  follows : 
To  5  c.c.  of  the  inulin  solution  in  a  test-tube  add  one  drop  of  concentrated 
hydrochloric  acid,  neutralize  with  concentrated  KOH  solution  and  test 
the  reducing  action  of  1  c.c.  of  the  resulting  solution  upon  1  c.c.  of  diluted 
(1:4)  Fehling's  solution.  This  will  show  the  normal  reducing  power 
of  the  inulin  solution.     In  case  the  inulin  was  hydrolyzed,  the  Fehling's 


POLYSACCHARIDES.  27 

LICHENIN,    (C0H10O.,)x. 

Lichenin  may  be  obtained  from  Cetraria  islandica  (  Iceland 
moss).  It  forms  a  difficultly  soluble  jelly  in  cold  water  and 
an  opalescent  solution  in  hot  water.  It  is  optically  inactive 
and  gives  no  color  with  iodine.  Upon  hydrolysis  with  dilute 
mineral  acids  lichenin  yields  dextrins  and  dextrose.  It  is  said 
to  be  most  nearly  related  chemically  to  starch.  Saliva,  pan- 
creatic juice,  malt  diastase  and  gastric  juice  have  no  noticeable 
action  on  lichenin. 

DEXTRIN,    (C6H10O5)x. 

The  dextrins  are  the  bodies  formed  midway  in  the  stages  of 
the  hydrolysis  of  starch  by  weak  acids  or  an  enzyme.  They 
are  amorphous  bodies  which  are  easily  soluble  in  water,  acids 
and  alkalis  but  are  insoluble  in  alcohol  or  ether.  Dextrins  are 
dextro-rotatory  and  are  not  fermentable  by  yeast. 

The  dextrins  may  be  hydrolyzed  by  dilute  acids  to  form  dex- 
trose. With  iodine  one  form  of  dextrin  (ervthro-dextrin) 
gives  a  red  color.  Their  power  to  reduce  Fehling's  solution 
is  questioned. 

Experiments  ox  Dextrix. 

1.  Solubility. — Test  the  solubility  of  pulverized  dextrin  in 
the  ordinary  solvents  (see  page  4). 

2.  Iodine  Test.- — Place  a  drop  of  dextrin  solution  in  one 
of  the  depressions  of  the  test-tablet  and  add  a  drop  of  a  dilute 
solution  of  iodine  in  potassium  iodide.  A  red  color  results. 
If  the  reaction  is  not  sufficiently  pronounced  make  a  stronger 
solution  from  the  pulverized  dextrin  and  repeat  the  test.  The 
solution  should  be  slightly  acid  to  secure  the  best  results. 

3.  Fehling's  Test. — See  if  the  dextrin  solution  will  reduce 
Fehling's  solution. 

4.  Hydrolysis  of  Dextrin. — Take  25  c.c.  of  dextrin  solu- 
tion in  a  small  beaker,  add  5  drops  of  dilute  HC1,  and  boil. 

test  in  the  hydrolysis  experiment  should  show  a  more  pronounced  reduction 
than  that  observed  in  the  check  experiment. 


28  PHYSIOLOGICAL    CHEMISTRY. 

By  means  of  a  small  pipette,  at  the  end  of  each  minute,  remove 
a  drop  of  the  solution  to  one  of  the  depressions  of  the  test- 
tablet  and  make  the  iodine  test.  The  power  of  the  solution 
to  produce  a  color  with  iodine  should  rapidly  disappear. 
When  a  negative  reaction  is  obtained  cool  the  solution  and 
neutralize  it  with  solid  KOH.  Try  Fehling's  test  (see  page 
8).  This  reaction  is  now  strongly  positive,  due  to  the  forma- 
tion of  a  reducing  sugar.  Determine  the  nature  of  the  sugar 
by  means  of  the  phenylhydrazin  test  (see  pages  5  and  6). 

5.  Influence  of  Tannic  Acid. — Add  an  excess  of  tannic 
acid  solution  to  a  small  amount  of  dextrin  solution  in  a  test- 
tube.  No  precipitate  forms.  This  result  differs  from  the 
result  of  the  similar  experiment  upon  starch  (see  Starch,  8, 
page  25). 

6.  Diffusibility  of  Dextrin. —  (See  Starch,  9,  page  25.) 

7.  Precipitation  by  Alcohol. — To  about  50  c.c.  of  95  per 
cent  alcohol  in  a  small  beaker  add  about  10  c.c.  of  a  concen- 
trated dextrin  solution.  Dextrin  is  thrown  out  of  solution  as 
a  gummy  white  precipitate.  Compare  the  result  with  that  ob- 
tained under  Dextrose,  5,  page  6. 

CELLULOSE,  (CeH10O5)x. 

This  complex  polysaccharide  forms  a  large  portion  of  the 
cell  wall  of  plants.  It  is  extremely  insoluble  and  its  molecule 
is  much  more  complex  than  the  starch  molecule.  The  best 
quality  of  filter  paper  and  the  ordinary  absorbent  cotton  are 
good  types  of  cellulose. 

Experiments  on  Cellulose. 

1.  Solubility. — Test  the  solubility  of  cellulose  in  the  ordi- 
nary solvents  (see  page  4). 

2.  Iodine  Test. — Add  a  drop  of  dilute  iodine  solution  to  a 
few  shreds  of  cotton  on  a  test-tablet.  Cellulose  differs  from 
starch  and  dextrin  in  giving  no  color  with  iodine. 

3.  Formation  of  Amyloid.1 — Add  10  c.c.  of  dilute  and  5 

'This  body  derives  its  name  from  amylum  (starch)  and  is  not  to  be 
confounded  with  amyloid,  the  gluco-proteid  (page  62). 


REVIEW    OF    CARBOHYDRATES. 


29 


c.c.  of  concentrated  H2S04  to  some  absorbent  cotton   in  a 

tot  tube.  When  entirely  dissolved  (without  heating)  pour 
one-half  of  the  solution  into  another  test-tube,  cool  it  and 
dilute  with  water.  Amyloid  forms  as  a  gummy  precipitate 
and  gives  a  brown  or  blue  coloration  with  iodine. 

After  allowing  the  second  portion  of  the  acid  solution  of 
cotton  to  stand  about  10  minutes  dilute  it  with  water  in  a 
small  beaker  and  boil  for  15-30  minutes.  Now  cool,  neutral- 
ize with  solid  KOH  and  test  with  Fehling's  solution.  Dex- 
trose has  been  formed  from  the  cellulose  by  the  action  of 
the  acid. 

4.  Schweitzer's    Solubility   Test. — Heat   some   absorbent 
cotton  in  a  test-tube  with  Schweitzer's  reagent.1     When  com 
pletely  dissolved  acidify  the  solution  with   acetic  acid.     An 
amorphous  precipitate  of  cellulose  is  produced.     Schweitzer's 
reagent  is  the  only  solvent  for  cellulose. 

REVIEW  OF  CARBOHYDRATES. 

In  order  to  facilitate  the  student's  review  of  the  carbo- 
hydrates, the  preparation  of  a  chart  similar  to  the  appended 


MODEL  CHART 

FOR    REVIEW 

PURPOSES. 

>% 

Carbohydrate. 

>< 

3 
"5 

8 

H 

V 

a 
'■S 
0 

V 

H 

"u 

0 
0 

•s 

H 

9 

s 

6 
? 

V 

H 

"so 

c 

2 

V 

H 

"u 

if 

1) 

H 

V 

-0 
a 
a 
>- 

H 
V 

H 

-0 

Molisch's 

Reaction. 

recipitation  b 

Alcohol. 

Osazon. 

Rotation. 

D  ffusibility 

I 

c 

1 
u 

Remarks. 

H 

b. 

« 

55 

aa 

Oh 



Dextrose. 

Maltose. 

Lactose. 

Saccharose. 
Starch. 

— 

Inulin. 

Dextrin. 

Cellulose. 

1  Schweitzer's  reagent  is  made  by  adding  potassium  hydroxide  to  a 
solution  of  cupric  sulphate  which  contains  some  ammonium  chloride.  A 
precipitate  of  cupric  hydroxide  forms  and  this  is  filtered  off,  washed  and 
brought  into  solution  in  20  per  cent  ammonium  hydroxide. 


30  PHYSIOLOGICAL    CHEMISTRY. 

model  is  recommended.  The  signs  -f-  and  —  may  be  con- 
veniently used  to  indicate  positive  and  negative  reaction. 
Only  those  carbohydrates  which  are  of  greatest  importance 
from  the  standpoint  of  physiological  chemistry  have  been  in- 
cluded in  the  chart. 

"  Unknown  "  Solutions  of  Carbohydrates. 

At  this  point  the  student  will  be  given  several  "  unknown  " 
solutions,  each  solution  containing  one  or  more  of  the  carbohy- 
drates studied.  He  will  be  required  to  detect,  by  means  of  the 
tests  on  the  preceding  pages,  each  carbohydrate  constituent  of 
the  several  "  unknown  "  solutions  and  hand  in,  to  the  instruc- 
tor, a  written  report  of  his  findings,  on  slips  furnished  by  the 
laboratory. 

The  scheme  given  on  page  31  may  be  of  use  in  this  con- 
nection. 


REVIEW    OF    CARBOHYDRATES. 


31 


c 

0 

(« 

a. 

0 

Ih 

■O 

£ 

u 

«*H 

in 

cs 

w 

H 

r, 

< 
P< 

<L> 

H 

Q 

>H 

d 

ffi 

x 

O 

PQ 

Xj 

Pi 
< 

'£ 

u 

."2 

"S 

fe 

rt 

O 

J>, 

J5 

.5 

O 

1— 1 

H 

U 

rt 

w 

E 

H 

u. 

W 

0 

Q 

<u 

N 

W 

"c5 

X 

Im 

H 

a 

u 

Pi 

c 

O 

<u 

fe 

_c 

w 


w 

wi    3 

K 

•-    O 

U 

3  -~ 
0  3 

t/j 

5  0 

3     !/J 

O    oj 

2  — 

£  0 

•- 


H 


u 

.2  r,  ~  n  0 

2 

7 

0 
0. 

'  solut 

f  conci 

heat 

neutr 

soluti 

u 

O  TJ  —    <U 

5 

'  unknown 
one  drop  1 
z   acid    an 
uite.     Coo 
hydroxid 
's  test. 

u 

rt 

u 

■3 

Q 

S 

q 

•r-=  p  ao 
<u  *c  b  3  5  3 

«J 

—  ~  -s  c.2a 

0 

u 

a 
c 

1 

:.c.  of 
-tube 
ydrocl 
or  one 
potas: 
by  Fel 

V 

-j 

w    -jr  aG  "*■'    r- 

1  "^  3  A 

•—3  </> 

5  n  U  '_ 
.2-3  'r  - 
*j   rt  P  B 

3  '->  re  2 

~  S  ~  o  3: 
K  «  «|    . 

-    -   7  .     c 

►2  re      •   .2 

'-'  ■—  w  *" 

■Sof  ^ 


.J.  °  "3 


u    3 


p  o  3  re  3 
■**■**  i-  a  -    43 

KOU        3 


Z  1; 


10  <u         hr  —   '" 
f2  re£=  -~ 

B     go     «^ 

•5  *3-Q.N    - 


c 

O   «i> 
•-    "> 


2  .3  E-= 
re  re  P. 


Ph£  « 


«  2 


pq 


H 


2  ~ 


oS5 


Z  ta 


1   T3    1)    u-    3 

^  rt-5  °  2 

re  *^      #^ 
a>  „         3  re 

rt   £    > ,  n    C 

•_n  o  _o  re  o> 

P  •*-        u  3 

*>      £  33  vm 
te  c  §  >. 

Q-—    <u  ■*.    3 

^  O   i;    w  (/1 

'  =  -tr  JZ   <y 

*-•  »>    O.  <_.  +j 


«  5 


rt 


Ph  o 


- 


O  -  Q< 

b;   C  O 

^  2  re 

M  * 


•—    3    u 

.a  -p  re 

i:  ""  « 
■-       c 

h  o  re 

n: 

ty  3  5  ■ 


c 

i>2§ 

u 

Ih 

60  «tj 

•3 

3    W    Ifl 

<L) 

rt  : 

1-  "O    > 

■^  rt  3 

O 

X  S  0 

U  —    3 

rt 

-0      J5t 

•  2  3 

3    c/i    3 

<u  a  « 

0^3" 

rt 

&        <u 

u 

■—  "3  — 

a> 

^-3  3 

'-^ 

—    rt  «j_ 

O 

.2  S  2 
%  hop 

'~ 

.300 

*-.    U    en 

U 

-,, 

bo 

0 

i. 

-. 

Ph  a 


z  -p 


p  >^ 

.3t3 


CHAPTER   II. 

SALIVARY    DIGESTION. 

The  saliva  is  secreted  by  three  pairs  of  glands,  the  sub- 
maxillary, sublingual  and  parotid,  reinforced  by  numerous 
small  glands  called  buccal  glands.  The  saliva  secreted  by 
each  pair  of  glands  possesses  certain  definite  characteristics 
peculiar  to  itself.  For  instance,  in  man,  the  parotid  glands 
ordinarily  secrete  a  thin,  watery  fluid,  the  submaxillary  glands 
secrete  a  somewhat  thicker  fluid  containing  mucin,  while  the 
product  of  the  sublingual  glands  has  a  more  mucilaginous 
character.  The  saliva  as  collected  from  the  mouth  is  the  com- 
bined product  of  all  the  glands  mentioned. 

The  saliva  may  be  induced  to  flow  by  many  forms  of  stimuli, 
such  as  chemical,  mechanical,  electrical,  thermal  and  psychical, 
the  nature  and  amount  of  the  secretion  depending,  to  a  limited 
degree,  upon  the  particular  class  of  stimuli  employed  as  well 
as  upon  the  character  of  the  individual  stimulus.  For  example, 
in  experiments  upon  dogs  it  has  been  found  that  the  mechanical 
stimulus  afforded  by  dropping  several  pebbles  into  the  animal's 
mouth  caused  the  flow  of  but  one  or  two  drops  of  saliva, 
whereas  the  mechanical  stimulus  afforded  by  sand  in  the  mouth 
induced  a  copious  flow  of  a  thin  watery  fluid.  Again,  when 
ice-water  or  snow  was  placed  in  the  animal's  mouth  no  saliva 
was  seen,  while  an  acid  or  anything  possessing  a  bitter  taste, 
which  the  dog  wished  to  reject,  caused  a  free  flow  of  the  thin 
saliva.  On  the  other  hand,  when  articles  of  food  were  placed 
in  the  dog's  mouth  the  animal  secreted  a  thicker  saliva  hav- 
ing a  higher  mucin  content — a  fluid  which  would  lubricate 
the  food  and  assist  in  the  passage  of  the  bolus  through  the 
oesophagus.  It  was  further  found  that  by  simply  drawing  the 
attention  of  the  animal  to  any  of  the  substances  named  above. 

32 


SALIVARY    DIGESTION.  33 

results  were  obtained  similar  to  those  secured  when  the  sub- 
stances were  actually  placed  in  the  animal's  mouth.  For  ex- 
ample, when  a  pretense  was  made  of  throwing  sand  into  the 
dog's  mouth,  a  watery  saliva  was  secreted,  whereas  food  under 
the  same  conditions  excited  a  thicker  and  more  slimy  secretion. 
The  exhibition  of  dry  food,  in  which  the  dog  had  no  particular 
interest  (dry  bread)  caused  the  secretion  of  a  large  amount 
of  saliva,  while  the  presentation  of  moist  food,  which  was 
eagerly  desired  by  the  animal,  called  forth  a  much  smaller 
secretion.  These  experiments  show  it  to  be  rather  difficult  to 
differentiate  between  the  influence  of  physiological  and  psy- 
chical stimuli. 

The  amount  of  saliva  secreted  by  an  adult  in  24  hours  has 
been  variously  placed,  as  the  result  of  experiment  and  obser- 
vation, between  1000  and  1500  c.c,  the  exact  amount  de- 
pending, among  other  conditions,  upon  the  character  of  the 
food. 

The  saliva  ordinarily  has  a  weak,  alkaline  reaction  to 
litmus,  but  becomes  acid  2-3  hours  after  a  meal  or  during 
fasting.  The  alkalinity  is  due  principally  to  di-sodium  hy- 
drogen phosphate  (Na2HP04)  and  its  average  alkalinity  may 
be  said  to  be  equivalent  to  0.08  — 0.1  per  cent  sodium  carbo- 
nate. The  saliva  is  the  most  dilute  of  all  the  digestive  fluids, 
having  an  average  specific  gravity  of  1.005  and  containing  only 
0.5  per  cent  of  solid  matter.  Among  the  solids  are  found  al- 
bumin, globulin,  mucin,  urea,  the  enzyme  ptyalin,  phosphates 
and  other  inorganic  constituents.  Potassium  sulphocyanide, 
KSCN,  is  also  generally  present  in  the  saliva.  It  has  been 
claimed  that  this  substance  is  present  in  greatest  amount  in 
the  saliva  of  habitual  smokers.  The  significance  of  sulpho- 
cyanide in  the  saliva  is  not  known;  it  may  come  from  the 
breaking  down  of  proteid. 

The  so-called  tartar  formation  on  the  teeth  is  composed  al- 
most entirely  of  calcium  phosphate  with  some  calcium  carbo- 
nate, mucin,  epithelial  cells  and  organic  debris  derived  from 
the  food.    The  calcium  salts  are  held  in  solution  as  acid  salts, 

4 


34  PHYSIOLOGICAL    CHEMISTRY. 

and  are  probably  precipitated  by  the  ammonia  of  the  breath. 
The  various  organic  substances  just  mentioned  are  carried 
down  in  the  precipitation  of  the  calcium  salts. 

The  saliva  contains  an  enzyme  known  as  ptyalin.  This  is 
an  amylolytic  enzyme,  so-called  because  it  possesses  the  prop- 
erty of  transforming  complex  carbohydrates  such  as  starch 
and  dextrin  into  simpler  bodies.  The  so-called  ferments  were 
formerly  divided  into  two  general  groups,  (i)  true  ferments 
or  so-called  organized  ferments  such  as  yeast  and  certain  bac- 
teria, which  were  supposed  to  act  by  virtue  of  vital  processes ; 
and  (2)  enzymes  such  as  ptyalin,  which  are  non-living,  un- 
organized bodies  of  a  chemical  nature.  Recently  this  distinc- 
tion between  true  ferments  and  enzymes  has  been  proven  to 
be  incorrect  since  it  has  been  shown  that  certain  of  the  bodies 
formerly  supposed  to  derive  their  ferment  activity  by  virtue  of 
their  vital  processes  in  reality  secrete  certain  definite  enzymes 
which  are  solely  responsible  for  their  ferment  activity.  In  no 
sense  is  it  a  vital  process  since  the  ferment  activity  is  entirely 
independent  of  the  vital  processes  of  the  cell.  We  may  define 
an  enzyme  as  an  unorganized,  soluble  ferment  which  is  elabor- 
ated by  an  animal  or  vegetable  cell  and  whose  activity  is  en- 
tirely independent  of  any  of  the  life  processes  of  such  a  cell. 

The  more  important  enzymes  may  be  classified,  according 
to  the  character  of  their  action,  as  follows:  (1)  amylolytic 
(starch  transforming),  (2)  proteolytic  (proteid  transform- 
ing), (3)  adipolytic  or  lipolytic  (fat  splitting),  (4)  inverting 
(possesses  inverting  power),  (5)  oxidative  (possesses  oxidiz- 
ing power),  and  (6)  proteid  coagulating. 

The  action  of  ptyalin  is  one  of  hydrolysis  and  through  this 
action  a  series  of  simpler  bodies  are  formed  from  the  complex 
starch.  The  first  product  of  the  action  of  the  ptyalin  of  the 
saliva  upon  starch  paste  is  soluble  starch  (amidulin)  and  its 
formation  is  indicated  by  the  disappearance  of  the  opalescence 
of  the  starch  solution.  This  body  resembles  true  starch  in 
giving  a  blue  color  with  iodine.  Next  follows  the  formation, 
in  succession,  of  a  series  of  dextrins,  called  erythro-dextrin, 


SALIVARY    DIGESTION.  35 

achroo-dextrin  and  malto-dextrin,  the  erythro-dextrin  being 
formed  directly  from  the  soluble  starch  and  later  being  itself 
transformed  into  achroo-dextrin  from  which  in  turn  is  pro- 
duced malto-dextrin.  Accompanying  each  dextrin  a  small 
amount  of  maltose  is  funned,  the  quantity  of  maltose  growing 
gradually  larger  as  the  process  of  transformation  progre 
Erythro  dextrin  gives  a  w<\  color  with  iodine,  the  other  dex- 
trins  give  no  color.  The  next  stage  is  the  transformation  of 
the  malto-dextrin  into  maltose  the  latter  being  the  principal 
end-pmduct  of  the  salivary  digestion  of  starch.  At  this  point 
small  amounts  of  iso-maltose  and  dextrose  are  formed  from 
the  maltose,  the  dextrose  being  produced  through  the  action 
of  the  enzyme  maltose. 

Ptyalin  acts  in  alkaline  or  neutral  solutions.  It  will  also  act 
in  the  presence  of  relatively  strong  combined  HO  (see  page 
84),  whereas  a  trace  (0.003  Per  cent  t()  0006  per  cent)  of 
ordinary  free  hydrochloric  acid  will  not  only  prevent  the  ac- 
tion but  will  destroy  the  enzyme.  By  sufficiently  increasing  the 
alkalinity  of  the  saliva  the  action  of  the  ptyalin  is  inhibited. 
It  has  recently  been  shown,  by  Cannon,  to  be  strongly  probable 
that  salivary  digestion  may  proceed  for  a  considerable  period 
after  the  food  reaches  the  stomach,  owing  to  the  slowness 
with  which  the  contents  are  thoroughly  mixed  with  the  acid 
gastric  juice  and  the  consequent  tardy  destruction  of  the 
enzyme. 

Microscopical  examination  of  the  saliva  reveals  salivary 
corpuscle^,  bacteria,  food  debris,  epithelial  cells,  mucus  and 
fungi.  In  certain  pathological  conditions  of  the  mouth,  pus 
cells  and  blood  corpuscles  may  be  found  in  the  saliva. 

Experiments  on  Saliva. 

A  satisfactory  method  of  obtaining  the  saliva  necessary  for 
the  experiments  which  follow  is  to  chew  a  small  piece  of  pure 
paraffin  wax  thus  stimulating  the  flow  of  the  secretion,  which 
may  be  collected  in  a  small  beaker.  Filtered  saliva  is  to  be 
used  in  every  experiment  except  for  the  microscopical  ex- 
amination. 


36 


PHYSIOLOGICAL    CHEMISTRY. 


1.  Microscopical  Examination. — Examine  a  drop  of  un- 
filtered  saliva  microscopically  and  compare  with  Fig.  18  below. 

2.  Reaction. — Test  the  reaction  to  litmus. 

Fig.  i  8. 


*  i  •*?>'& 


Microscopical    Constituents    of    Saliva. 

a,  Epithelial  cells ;  b,  salivary  corpuscles ;  c,  fat  drops ;  d,  leucocytes ;  e,  f  and 

g,    bacteria ;    h,   i    and    k,    fission-fungi. 

3.  Specific  Gravity. — Partially  fill  a  urinometer  cylinder 
with  saliva,  introduce  the  urinometer  (see  Fig.  83,  page  232), 
and  observe  the  reading. 

4.  Test  for  Mucin. — To  a  small  amount  of  saliva  in  a  test- 
tube  add  1-2  drops  of  dilute  acetic  acid.    Mucin  is  precipitated. 

5.  Biuret  Test.1 — Render  a  little  saliva  alkaline  with  an 
equal  volume  of  KOH  and  add  a  few  drops  of  a  very  dilute 
(2-5  drops  in  a  test-tube  of  water)  cupric  sulphate  solution. 
The  formation  of  a  purplish-violet  color  is  due  to  mucin. 

6.  Millon's  Reaction.2 — Add  a  few  drops  of  Millon's  re- 
agent to  a  little  saliva.  A  light  yellow  precipitate  formed  by 
the  mucin  gradually  turns  red  upon  being  gently  heated. 

7.  Preparation  of  Mucin. — Pour  15  c.c.  of  saliva  into  100 
c.c.  of  95  per  cent  alcohol,  stirring  constantly.  Cover  the 
vessel  and  allow  the  precipitate  to  stand  at  least  12  hours. 
Pour  off  the  supernatant  liquid,  collect  the  precipitate  on  a 
filter  and  wash  it,  in  turn,  with  alcohol  and  ether.  Finally  dry 
the  precipitate,  remove  it  from  the  paper  and  make  the  follow- 
ing tests  on  the  mucin:  (a)  Test  its  solubility  in  the  ordinary 

1  The  significance  of  this  reaction  is  pointed  out  on  page  45. 

2  The  significance  of  this  reaction  is  pointed  out  on  page  44. 


SALIVARY    DIGESTION.  37 

solvents  (see  page  4),  ( /> )  Millon's  reaction,  (c)  dissolve 
a  small  amount  in  KOH,  and  try  the  biuret  test  on  the  solution, 
(d)  boil  the  remainder,  with  10-25  c.c.  of  water  to  which  5 
c.c.  of  dilute  I1C1  has  been  added,  until  the  solution  becomes 
brownish.  Cool,  render  alkaline  with  solid  KOH,  and  test  by 
Fehling's  solution.  A  reduction  should  take  place.  Mucin 
is  what  is  known  as  a  compound  proteid  or  glucoproteid  (see 
p.  6] )  and  upon  boiling  with  the  acid  the  carbohydrate  group 
in  the  molecule  has  been  split  off  from  the  proteid  portion  and 
its  presence  is  indicated  by  the  reduction  of  Fehling's  solution. 

8.  Inorganic  Matter. — Test  for  chlorides,  phosphates, 
sulphates  and  calcium.  For  chlorides,  acidify  with  HN03  and 
add  AgNOa.  For  phosphates,  acidify  with  HXOa,  heat  and 
add  molybdic  solution.1  For  sulphates,  acidify  with  HC1  and 
add  BaCl2  and  warm.  For  calcium,  acidify  with  acetic  acid, 
CH:iCOOH.  and  add  ammonium  oxalate,   (NH4)2C204. 

9.  Filtration  Experiment. — Place  filter  papers  in  two  fun- 
nels, and  to  each  add  an  equal  quantity  of  starch  paste  (5  c.c). 
Add  a  few  drops  of  saliva  to  one  lot  of  paste  and  an  equivalent 
amount  of  water  to  the  other.  Note  the  progress  of  filtration 
in  each  case.  Why  does  one  solution  filter  more  rapidly  than 
the  other? 

10.  Test  for  Nitrites. — Add  1-2  drops  of  dilute  H2S04  to 
a  little  saliva  and  thoroughly  stir.  Now  add  a  few  drops  of- a 
potassium  iodide  solution  and  some  starch  paste.  Nitrous 
acid  is  formed  which  liberates  iodine  causing  the  formation 
of  the  blue  iodide  of  starch. 

11.  Sulphocyanide  Tests. —  (a)  Ferric  Chloride  Test. — 
To  a  little  saliva  in  a  small  porcelain  crucible,  or  dish,  add  a 
few  drops  of  dilute  ferric  chloride  and  acidify  slightly  with 
HC1.  Red  ferric  sulphocyanide  forms.  To  show  that  the  red 
coloration  is  not  due  to  iron  phosphate  add  a  drop  of  HgCl2 
when  colorless  mercuric  sulphocyanide  forms. 

1  Molybdic  solution  is  prepared  as  follows,  the  parts  being  by  weight : 
1  part,  molybdic  acid. 

4  parts,  ammonium  hydroxide   (Sp.  gr.  0.96). 
15  parts,   nitric  acid    (Sp.  gr.    1.2). 


38  PHYSIOLOGICAL    CHEMISTRY. 

(b)  Solera's  Reaction. — This  test  depends  upon  the  libera- 
tion of  iodine  through  the  action  of  sulphocyanide  upon  iodic 
acid.  Moisten  a  strip  of  starch  paste-iodic  acid  test  paper1 
with  a  little  saliva.  If  sulphocyanide  be  present  the  test  paper 
will  assume  a  blue  color,  due  to  the  liberation  of  iodine  and  its 
subsequent  formation  of  the  so-called  iodide  of  starch. 

12.  Digestion  of  Starch  Paste. — To  25  c.c.  of  starch  paste 
in  a  small  beaker,  add  5  drops  of  saliva  and  stir  thoroughly. 
At  intervals  of  a  minute  remove  a  drop  of  the  solution  to  one 
of  the  depressions  in  a  test-tablet  and  test  by  the  iodine  test. 
If  the  blue  color  with  iodine  still  forms  after  5  minutes,  add 
another  5  drops  of  saliva.  The  opalescence  of  the  starch  solu- 
tion should  soon  disappear,  indicating  the  formation  of  sol- 
uble starch  which  gives  a  blue  color  with  iodine.  This  body 
should  soon  be  transformed  into  erythro-dextrin  which  gives  a 
red  color  with  iodine  and  this  in  turn  should  pass  into  achroo- 
dextrin  which  gives  no  color  with  iodine.  This  is  called  the 
achromic  point.  When  this  point  is  reached  test  by  Fehling's 
test  to  show  the  production  of  a  reducing  body.  A  body 
formed  coincidently  with  erythro-dextrin  may  yield  a  slight 
response  to  Fehling's  test.  What  body  is  it?  How  long  did 
it  take  for  a  complete  transformation  of  the  starch? 

13.  Digestion  of  Dry  Starch. — In  a  test-tube  shake  up  a 
small  amount  of  dry  starch  with  a  little  water.  Add  a  few 
drops  of  saliva,  mix  well  and  allow  to  stand.  After  10-20 
minutes  filter  and  test  the  filtrate  by  Fehling's  test.  What  is 
the  result  and  why  ? 

14.  Digestion  of  Inulin. — To  5  c.c.  of  inulin  solution  in  a 
test-tube  add  10  drops  of  saliva  and  place  the  tube  in  the 
water-bath  at  400  C.  After  one-half  hour  test  the  solution  by 
Fehling's  test.2  Is  any  reducing  substance  present?  What 
do  you  conclude  regarding  the  salivary  digestion  of  inulin? 

1  This  test  paper  is  prepared  as  follows :  Saturate  a  good  quality  of 
filter  paper  with  0.5  per  cent  starch  paste  containing  a  little  iodic  acid 
and  allow  the  paper  to  dry  in  the  air.  Cut  it  in  strips  of  suitable  size 
and  preserve  for  use. 

*  If  the  inulin  solution  gives  a  reduction  before  being  acted  upon  by  the 
saliva  it  will  be  necessary  to  determine  the  extent  of  this  original  reduc- 
tion by  means  of  a  "check"  test  (see  page  26). 


SALIVARY    DIGESTION.  39 

15.  Influence  of  Temperature. — In  each  of  four  tubes 
place  about  5  c.c.  of  starch  pa^te.  Immerse  one  tube  in  cold 
water  from  the  faucet,  keep  a  second  at  room  temperature  and 
place  a  third  <>n  the  water-bath  at  40  ('.  Now  add  to  the  con- 
tents of  each  of  these  three  tubes  two  drops  of  saliva  and  shake 
well :  to  the  contents  of  the  fourth  tube  add  two  drops  of  boiled 
saliva.  Test  frequently  by  the  iodine  tot.  using  the  test- 
tablet,  ami  note  in  which  tube  the  most  rapid  digestion  occurs. 
Explain  the  results. 

16.  Influence  of  Dilution. — Take  a  series  of  6  test-tubes 
each  containing  9  c.c.  of  water.  Add  1  c.c.  of  saliva  to  tube  1 
and  shake  thoroughly.  Remove  1  c.c.  of  the  solution  from 
tube  1  to  tube  2  and  after  mixing  thoroughly  remove  1  c.c. 
from  tube  2  to  tube  3.  Continue  in  this  manner  until  you  have 
6  saliva  solutions  of  gradually  decreasing  strength.  Now  add 
starch  paste  in  equal  amounts  to  each  tube,  mix  very  thor- 
oughly and  place  on  the  water-bath  at  400  C.  After  10-20 
minutes  test  by  both  the  iodine  and  Fehling's  tests.  In  how 
great  dilution  does  your  saliva  act? 

17.  Influence  of  Acids  and  Alkalis. —  (a)  Influence  of  Free 
Acid. — Prepare  a  series  of  6  tubes  in  each  of  which  is  placed 
4  c.c.  of  one  of  the  following  strengths  of  free  II CI :  0.2  per 
cent.  o.  1  per  cent.  0.05  per  cent,  0.025  per  cent,  0.0125  per  cent 
and  0.006  per  cent.  Xow  add  2  c.c.  of  starch  paste  to  each 
tube  and  shake  them  thoroughly.  Complete  the  solutions  by 
adding  2  c.c.  of  saliva  to  each  and  repeat  the  shaking.  The 
total  acidity  of  this  series  would  be  as  follows:  0.1  per  cent, 
0.05  per  cent.  0.025  per  cent.  0.0125  per  cent,  0.006  per  cent 
and  0.003  Per  cent-  Place  these  tubes  on  the  water-bath  at 
40  C.  for  10-20  minutes.  Divide  the  contents  of  each  tube 
into  two  parts,  testing  one  part  by  the  iodine  test  and  testing 
the  other,  after  neutralization,  by  Fehling's  test.  What  do 
you  rind  ? 

(b)  Influence  of  Combined  Acid. — Repeat  the  first  three 
experiments  of  the  above  series  using  combined  hydrochloric 
acid  1  see  page  84)  instead  of  the  free  acid.  How  does  the 
action  of  the  combined  acid  differ  from  that  of  the  free  acid/ 


40  PHYSIOLOGICAL    CHEMISTRY. 

(c)  Influence  of  Alkali. — Repeat  the  first  four  experiments 
under  (a)  replacing  the  HC1  by  2  per  cent,  1  per  cent,  0.5 
per  cent  and  0.25  per  cent  Na2C03.  Neutralize  the  alkalinity 
before  trying  the  iodine  test  (see  Starch,  5,  page  24). 

(d)  Nature  of  the  Action  of  Acid  and  Alkali. — Place  2 
c.c.  of  saliva  and  2  c.c.  of  0.2  per  cent  HC1  in  a  test-tube  and 
leave  for  15  minutes.  Neutralize  the  solution,  add  4  c.c.  of 
starch  paste  and  place  the  tube  on  the  water-bath  at  40  °  C. 
In  10  minutes  test  by  the  iodine  and  Fehling's  tests  and  ex- 
plain the  result.  Repeat  the  experiment  replacing-  the  0.2  per 
cent  HC1  by  2  per  cent  Na2COs.  What  do  you  deduce  from 
these  two  experiments? 

18.  Influence  of  Metallic  Salts,  etc. — In  each  of  a  series 
of  tubes  place  4  c.c.  of  starch  paste  and  ^  c.c.  of  one  of  the 
solutions  named  below.  Shake  well,  add  y2  c.c.  of  saliva  to 
each  tube,  thoroughly  mix,  and  place  on  the  water-bath  at 
400  C.  for  10-20  minutes.  Show  the  progress  of  digestion 
by  means  of  the  iodine  and  Fehling  tests.  Use  the  following 
chemicals :  Metallic  salts,  10  per  cent  plumbic  acetate,  2  per 
cent  cupric  sulphate,  5  per  cent  ferric  chloride,  8  per  cent 
mercuric  chloride;  Neutral  salts,  10  per  cent  sodium  chloride, 
3  per  cent  barium  chloride,  10  per  cent  Rochelle  salt.  Also 
try  the  influence  of  2  per  cent  carbolic  acid,  95  per  cent 
alcohol,  and  ether  and  chloroform.  What  are  your  con- 
clusions? 

19.  Excretion  of  Potassium  Iodide. — Ingest  a  small  dose 
of  potassium  iodide  (0.2  gram)  contained  in  a  gelatin  cap- 
sule, quickly  rinse  out  the  mouth  with  water  and  then 
test  the  saliva  at  once  for  iodine.  This  test  should  be 
negative.  Make  additional  tests  for  iodine  at  2  minute 
intervals.  The  test  for  iodine  is  made  as  follows :  Take 
1  c.c.  of  NaN02  and  1  c.c.  of  dilute  H2SCV  in  a  test- 
tube,  add  a  little  saliva  directly  from  the  mouth,  and  a  small 
amount  of  starch  paste.     If  convenient,  the  urine  may  also 

1  Instead  of  this  mixture  a  few  drops,  of  HN03  possessing  a  yellowish 
or  brownish  color  due  to  the  presence  of  HNO2  may  be  employed. 


SALIVARY    DIGESTION.  4 1 

be  tested.  The  formation  of  a  blue  color  signifies  that  the 
potassium  iodide  is  being  excreted  through  the  salivary  glands. 
Note  the  length  of  time  elapsing  between  the  ingestion  of  the 
potassium  iodide  and  the  appearance  of  the  first  traces  of  the 
substance  in  the  saliva.  The  chemical  reactions  taking  place 
in  this  experiment  are  indicated  in  the  following  equations : 

(a)  2NaN02  +H2S04  =  2HN02  +  Na2S04. 

(b)  2KI  +  H2S04  =  2HI  +  K2S04. 

(c)  2HN02+2HI  =  I2  +  2H20  +  2NO. 

20.  Qualitative  Analysis  of  the  Products  of  Salivary 
Digestion. — To  25  c.c.  of  the  products  of  salivary  digestion 
(saved  from  Experiment  12  or  furnished  by  the  instructor), 
add  100  c.c.  of  95  per  cent  alcohol.  Allow  to  stand  until  the 
white  precipitate  has  settled.  Filter,  evaporate  the  filtrate 
to  dryness,  dissolve  the  residue  in  5-10  c.c.  of  water  and  try 
Fehling's  test  (page  8)  and  the  phenylhydrazin  reaction  (see 
Dextrose,  3,  page  5).  On  the  dextrin  precipitate  try  the  iodine 
test  (page  24).  Also  hydrolyze  the  dextrin  as  given  under 
Dextrin,  4,  page  27. 


CHAPTER    III. 

PROTEIDS. 

Proteids  are  a  group  of  very  complex  organic  substances, 
constituting  the  most  important  class  of  food  stuffs  and  are 
widely  distributed  in  animal  and  vegetable  tissues.  Every 
proteid  contains  carbon,  hydrogen,  nitrogen,  oxygen  and  sul- 
phur, and  a  relatively  small  number  contain  phosphorus  and 
iron  in  addition.  The  percentage  composition  of  the  more 
important  members  of  the  group  would  fall  within  the  fol- 
lowing limits:  C  (51  per  cent  to  55  per  cent),  H  (6  per  cent 
to  7.3  per  cent),  N  (15  per  cent  to  19  per  cent),  O  (21  per 
cent  to  23  per  cent),  S  (0.3  per  cent  to  2.5  per  cent),  and  P 
(0.4  per  cent  to  0.8  per  cent  when  present)  :  Fe  occurs  only 
in  traces.  The  most  important  element  of  the  proteid  mole- 
cule is  the  nitrogen.  The  human  body  needs  nitrogen  for  the 
continuation  of  life,  but  it  cannot  use  the  nitrogen  of  the  air 
or  that  in  various  other  combinations  such  as  we  find  in 
nitrites,  etc.  However,  in  the  proteid  molecule  the  nitrogen 
is  present  in  a  form  which  is  utilizable  by  the  body. 

No  definite  knowledge  has  yet  been  secured  regarding  the 
constitutional  formula  or  the  molecular  weight  of  proteid 
material.  The  molecular  weight  of  tgg  albumin  has  been 
placed  at  about  15,000  and  the  formula  for  the  crystallized 
product  has  been  calculated  as  C239H3S6N58S207S.  Many  im- 
portant and  valuable  investigations  have  been  promoted  re- 
cently on  the  subject  of  the  constitution  of  the  proteid  mole- 
cule and  our  knowledge  has  been  largely  increased. 

The  proteids  may  be  classified  as  follows : 

I.    SIMPLE   PROTEIDS. 

1.    NATIVE  SIMPLE  PROTEIDS. 

(a)  Albumins — egg  albumin,  serum  albumin  and  vege- 
table albumins. 

42 


PROTEIDS.  43 

( b)  Globulins — scrum  globulin,  ovoglobulin,  edestin  and 
other  vegetable  globulins. 

(c)  Phospho-proteids  (nucleo-albumins)  caseinogen 
and  vitellin. 

2.    DERIVED  SIMPLE  PROTEIDS. 

(a)  Albuminates — acid  albuminate  and  alkali  albuminate. 

I /> )  Proteoses  (or  albumoses )  and  peptones — proto- 
proteose,  heteyoproteose  and  deuteroproteose;  amphopeptone 
and  antipeptone. 

(c)  Coagulated  Proteids — fibrin,  and  the  products  of  heat 
coagulation,  etc. 

II.    COMPOUND  PROTEIDS. 

(a)  Glucoproteids — mucins  (from  fluids  and  secretions); 
mucoids,  c.  g.,  osseomucoid  and  iendomucoid ;  amyloid. 

(b)  Nucleo-proteids. 

(c)  Haemoglobins. 

III.    ALBUMINOIDS,  ALBUMOIDS  OR  PRO- 
TEOIDS  (PROTEID-LIKE  BODIES). 

(a)  Chondroalbumoid — isolated  from  cartilage. 

(b)  Collagen — constituent  of  connective  tissue  and  par- 
ticularly abundant  in  tendinous  tissue. 

(  c)  Elastin — constituent  of  connective  tissue  and  particu- 
larly abundant  in  ligament. 

(d)  Gelatin — product  of  the  hydrolysis  of  collagen. 

(<?)  Keratin — forms  the  major  portion  of  hair,  hoof,  horn, 
etc. 

(/)  Osseoalbumoid — isolated  from  bone. 

(g)  Reticulin — found  in  fibers  of  reticular  tissue. 

GENERAL  COLOR  REACTIONS  OF  PROTEIDS. 

These  color  reactions  are  due  to  a  reaction  between  some 
one  or  more  of  the  constituent  radicals  or  groups  of  the  com- 
plex proteid  molecule  and  the  chemical  reagent  or  reagents 


44  PHYSIOLOGICAL    CHEMISTRY. 

used  in  any  given  test.  Not  all  proteids  contain  the  same 
groups  and  for  this  reason  the  various  color  tests  will  yield 
reactions  varying  in  intensity  of  color  according  to  the  nature 
of  the  groups  contained  in  the  particular  proteid  under  ex- 
amination. Various  substances  not  proteids  respond  to  cer- 
tain of  these  color  reactions  and  it  is  therefore  essential  to 
submit  the  material  under  examination  to  several  tests  before 
concluding  definitely  regarding  its  nature. 

TECHNIQUE  OF  THE  COLOR  REACTIONS. 

1.  Millon's  Reaction. — To  5  c.c.  of  a  dilute  egg  albumin 
solution  in  a  test-tube  add  a  few  drops  of  Millon's  reagent. 
A  white  precipitate  forms  which  turns  red  when  heated.  This 
test  is  a  particularly  satisfactory  one  for  use  on  solid  proteids, 
in  which  case  the  reagent  is  added  directly  to  the  solid  sub- 
stance and  heat  applied,  which  causes  the  substance  to  assume 
a  red  color. 

The  reaction  is  due  to  the  presence  of  the  hydroxy-phenyl 
group,  —  C6H4OH,  in  the  proteid  molecule  and  certain  non- 
proteids  such  as  tyrosin  and  phenol  (carbolic  acid)  also  re- 
spond to  the  reaction.  The  test  is  not  a  very  satisfactory  one 
for  use  in  solutions  containing  salts,  since  the  mercury  of  the 
Millon's  reagent1  is  thus  precipitated  and  the  reagent  rendered 
inert. 

2.  Xanthoproteic  Reaction. — To  2-3  c.c.  of  egg  albumin 
solution  in  a  test-tube  add  concentrated  HN03.  A  white  pre- 
cipitate forms,  which  upon  heating  turns  yellow  and  finally 
dissolves,  imparting  to  the  solution  a  yellow  color.  Cool  the 
solution  and  carefully  add  NH4OH  in  excess.  Note  that  the 
yellow  color  deepens  into  an  orange.  This  reaction  is  due  to 
the  presence  in  the  proteid  molecule  of  the  phenyl  group,  with 
which  the  nitric  acid  forms  certain  nitro  modifications.     The 

1  Millon's  reagent  consists  of  mercury  dissolved  in  nitric  acid  containing 
some  nitrous  acid.  It  is  prepared  by  digesting  one  part  (by  weight)  of 
mercury  with  two  parts  (by  weight)  of  HNOs  (sp.  gr.  1.42)  and  diluting 
the  resulting  solution  with  two  volumes  of  water. 


1'KOTEIDS.  45 

test  is  not  a  satisfactory  one  for  use  in  urinary  examination 
because  of  the  color  of  the  end-reaction. 

3.  Adamkiewicz  Reaction. — Thoroughly  mix  1  volume 
of  concentrated  1\LS04  and  2  volumes  of  acetic  acid  in  a  test- 
tube,  add  a  few  drops  of  egg  albumin  solution  and  heat  gently. 
A  reddish-violet  color  is  produced.  Gelatin  does  not  respond 
to  this  test.  This  reaction  shows  the  presence  of  the  trypto- 
phan group  (see  next  experiment).  The  test  depends  upon 
the  presence  of  glyoxylic  acid,  CHO*  COOI1  in  the  reagents. 
This  is  shown  by  the  failure  to  secure  a  positive  reaction  when 
acetic  acid  free  from  glyoxylic  acid  is  used. 

4.  Hopkins-Cole  Reaction. — Place  1-2  c.c.  of  egg  albumin 
solution  and  3  c.c.  of  glyoxylic  acid,  CHOCOOH,  solu- 
tion (Hopkins-Cole  reagent1)  in  a  test-tube  and  mix  thor- 
oughly. In  a  second  tube  place  5  c.c.  of  concentrated  sulphuric 
acid.  Incline  the  tube  containing  the  sulphuric  acid  and  by 
means  of  a  pipette  allow  the  albumin-glyoxylic  acid  solution  to 
flow  carefully  down  the  side.  When  stratified  in  this  manner 
a  reddish-violet  color  forms  at  the  zone  of  contact  of  the  two 
fluids.  This  color  is  due  to  the  presence  of  the  tryptophan 
group.  Gelatin  does  not  respond  to  this  test.  For  formula 
for  tryptophan  see  page  yy. 

5.  Biuret  Test. — To  2-3  c.c.  of  egg  albumin  solution  in 
a  test-tube  add  an  equal  volume  of  concentrated  KOH  solution, 
mix  thoroughly,  and  add  slowly  a  very  dilute  (2-5  drops  in  a 
test-tube  of  water)  cupric  sulphate  solution  until  a  purplish- 
violet  or  pinkish-violet  color  is  produced.  The  depth  of  the 
color  depends  upon  the  nature  of  the  proteid,  proteoses  and 
peptones  giving  a  decided  pink,  while  the  color  produced  with 
gelatin  is  not  far  removed  from  a  blue.  This  reaction  is  given 
by  those  bodies  which  contain  two  amino  groups  (CONHo, 
C(NH)NH2,  CH0NH0  or  CSNH2)  united  by  a  C  or  N  atom 
or  joined  together  directly,  therefore  certain  non-proteids  may 

1  Hopkins-Cole  reagent  is  prepared  as  follows :  To  one  liter  of  a  satu- 
rated solution  of  oxalic  acid  add  60  grams  of  sodium  amalgam  and  allow 
the  mixture  to  stand  until  the  evolution  of  gas  ceases.  Filter  and  dilute 
with  2-3  volumes  of  water. 


46  PHYSIOLOGICAL    CHEMISTRY. 

also  give  a  positive  biuret  reaction.  Proteid  material  responds 
positively  since  there  are  two  COXH2  groups  in  the  proteid 
molecule.  These  groups  are  found  in  a  substance  called 
biuret  (see  page  243)  which  may  be  formed  by  heating  urea 
to  1800  C. 

6.  Posner's  Modification  of  the  Biuret  Test. — This  test 
is  particularly  satisfactory  for  use  on  dilute  proteid  solutions, 
and  is  carried  out  as  follows :  To  some  dilute  egg  albu- 
min in  a  test-tube  add  one-half  its  volume  of  KOH  solution. 
Now  hold  the  tube  in  an  inclined  position  and  allow  some  very 
dilute  cupric  sulphate  solution,  made  as  suggested  on  page  45 
(5),  to  flow  down  the  side,  being  especially  careful  to  prevent 
the  fluids  from  mixing.  At  the  juncture  of  the  two  solutions 
the  typical  end-reaction  of  the  biuret  test  should  appear  as  a 
colored  zone  (see  Biuret  Test,  page  45). 

7.  Liebermann's  Reaction. — Add  about  10  drops  of  con- 
centrated egg  albumin  solution  (or  a  little  dry  egg  albumin) 
to  about  5  c.c.  of  concentrated  HC1  in  a  test-tube.  Boil  until 
a  pinkish-violet  color  results. 

PRECIPITATION  REACTIONS  AND   OTHER 
PROTEID  TESTS. 

There  are  three  forms  in  which  proteids  may  be  precipi- 
tated i.  e.,  unaltered,  as  an  albuminate,  and  as  an  insoluble  salt. 
An  instance  of  the  precipitation  in  a  native  or  unaltered  con- 
dition is  seen  in  the  so-called  salting-out  experiments.  Various 
salts,  notably  (NH4)2S04,  ZnS04,  MgS04,  Na2S04  and 
NaCl  possess  the  power,  when  added  in  solid  form  to  certain 
definite  proteid  solutions,  of  rendering  the  menstruum  inca- 
pable of  holding  the  proteid  in  solution,  thereby  causing  the 
proteid  to  be  precipitated  or  salted-out  to  use  the  common 
term.  Mineral  acids  and  alcohol  also  precipitate  proteids  un- 
altered. Proteids  are  precipitated  as  albuminates  when  treated 
with  certain  metallic  salts,  and  precipitated  as  insoluble  salts 
when  certain  weak  organic  acids  are  added  to  their  solutions. 


PROTEIDS.  47 

EXPERIMEN 

i.  Influence  of  Concentrated  Mineral  Acids,  Alkalis  and 
Organic  Acids. —  Prepare  5  test-tubes  each  containing  5  c.c. 
of  concentrated  egg  albumin  solution.  To  the  first  add  con- 
centrated H2S04,  drop  by  drop,  until  an  excess  of  the  acid  has 
been  added.  Note  any  changes  which  may  occur  in  the  solu- 
tion. Allow  the  tube  to  stand  for  24  hours  and  at  the  end  of 
that  period  observe  any  alteration  which  may  have  taken  place. 
Heat  the  tube  and  note  any  further  change  which  may  occur. 
Repeat  the  experiment  in  the  4  remaining  tubes  with  concen- 
trated HC1,  concentrated  1IXO...  concentrated  KOH  and 
CH3COOII.  How  do  strong  mineral  acids,  strong  alkali-  and 
strong  organic  acids  differ  in  their  action  toward  proteid  solu- 
tions? 

2.  Precipitation  by  Metallic  Salts. — Prepare  4  tubes  each 
containing  2-3  c.c.  of  dilute  egg  albumin  solution.  To  the  first 
add  mercuric  chloride,  drop  by  drop,  until  an  excess  of  the  re- 
agent has  been  added,  noting  any  changes  which  may  occur. 
Repeat  the  experiment  with  plumbic  acetate,  argentic  nitrate, 
cupric  sulphate,  ferric  chloride  and  barium  chloride. 

Egg  albumin  is  used  as  an  antidote  for  lead  or  mercury 
poisoning.     Why? 

3.  Precipitation  by  Alkaloidal  Reagents. — Prepare  6 
tubes  each  containing  2-3  c.c.  of  dilute  egg  albumin  solution. 
To  the  first  add  picric  acid  drop  by  drop  until  an  exce-s  of  the 
reagent  has  been  added,  noting  any  changes  which  may  occur. 
Repeat  the  experiment  with  trichloracetic  acid,  tannic  acid, 
phosphotungstic  acid,  plwspJwmolybdic  acid  and  potassio-mcr- 
curic  iodide.  Acidify  with  HC1  before  testing  with  the  three 
last  reagents. 

4.  Heller's  Ring  Test. — Place  5  c.c.  of  concentrated 
ll\03  in  a  test-tube,  incline  the  tube,  and  by  means  of  a 
pipette  allow  the  dilute  albumin  solution  to  flow  slowly  down 
the  side.1     The  liquids  should  stratify  with  the  formation  of 

1  An  apparatus  called  the  albumoscopc  has  been  devised  for  use  in  this 
test  and  has  met  with  considerable  favor. 


48  PHYSIOLOGICAL    CHEMISTRY. 

a  white  zone  of  precipitated  albumin  at  the  point  of  juncture. 
This  is  a  very  delicate  test  and  is  further  discussed  on  p.  290. 

5.  Roberts'  Ring  Test. — Place  5  c.c.  of  Roberts'  reagent1 
in  a  test-tube,  incline  the  tube,  and  by  means  of  a  pipette  allow 
the  albumin  solution  to  flow  slowly  down  the  side.  The 
liquids  should  stratify  with  the  formation  of  a  white  zone  of 
precipitated  albumin  at  the  point  of  juncture.  This  test  is  a 
modification  of  Heller's  ring  test  and  is  rather  more  satisfac- 
tory.     (See  page  291.) 

6.  Spiegler's  Ring  Test. — Place  5  c.c.  of  Spiegler's  re- 
agent2 in  a  test-tube,  incline  the  tube,  and  by  means  of  a 
pipette  allow  5  c.c.  of  albumin  solution,  acidified  with  acetic 
acid,  to  flow  slowly  down  the  side.  A  white  zone  will  form 
at  the  point  of  contact.  This  is  an  exceedingly  delicate  test, 
in  fact,  too  delicate  for  ordinary  clinical  purposes,  since  it 
serves  to  detect  albumin  when  present  in  the  merest  trace 
(1:250,000).     This  test  is  further  discussed  on  page  291. 

7.  Jolles'  Reaction. — Shake  5  c.c.  of  albumin  solution  with 
1  c.c.  of  30  per  cent  acetic  acid  and  4  c.c.  of  Jolles'  reagent3 
in  a  test-tube.  A  white  precipitate  of  albumin  should  form. 
Care  should  be  taken  to  use  the  correct  amount  of  acetic  acid. 
For  further  discussion  of  the  test  see  page  292. 

8.  Tanret's  Test. — To  5  c.c.  of  albumin  solution  in  a  test- 
tube  add  Tanret's  reagent,4  drop  by  drop,  until  a  turbidity  or 

1  Roberts'  reagent  is  composed  of  1  volume  of  concentrated  HNOs  and 
5  volumes  of  a  saturated  solution  of  MgSCX. 
"  Spiegler's  reagent  has  the  following  composition : 

Tartaric  acid   20  grams. 

Mercuric  chloride 40      " 

Glycerin    IOO       " 

Distilled  water    1000      " 

8  Jolles'  reagent  has  the  following  composition : 

Succinic  acid   40  grams. 

Mercuric  chloride 20      " 

Sodium  chloride   20      " 

Distilled  water    1000      " 

*  Tanret's  reagent  is  prepared  as  follows :  Dissolve  1.35  gram  of  mercuric 
chloride  in  25  c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium 
iodide  dissolved  in  25  c.c.  of  water,  then  make  the  total  solution  up  to  60  c.c. 
with  water  and  add  20  c.c.  of  glacial  acetic  acid  to  the  combined  solutions. 


PROTEIDS.  49 

precipitate  forms.  This  is  an  exceedingly  delicate  test.  Some- 
times the  albumin  solution  is  stratified  upon  the  reagent  as  in 
Heller's  or  Roberts'  ring  tests. 

9.  Sodium  Chloride  and  Acetic  Acid  Test.— Mix  two 
volumes  of  albumin  solution  and  1  volume  of  a  saturated  solu- 
tion of  sodium  chloride  in  a  test-tube,  acidify  with  acetic  acid 
and  heat  to  boiling.  The  production  of  a  cloudiness  or  the 
formation  of  a  precipitate  indicates  the  presence  of  albumin. 

10.  Acetic  Acid  and  Potassium  Ferrocyanide  Test. — To 
5  c.c.  of  dilute  egg  albumin  solution  in  a  test-tube  add  5-10 
drops  of  acetic  acid.  Mix  well,  and  add  potassium  ferrocy- 
anide, drop  by  drop,  until  a  precipitate  forms.  This  test  is 
very  delicate. 

11.  Salting-out  Experiments. —  (a)  To  25  c.c.  of  egg  albu- 
min solution  in  a  small  beaker  add  solid  (NH4)2S04  to  the 
point  of  saturation,  keeping  the  temperature  of  the  solution 
below  400  C.  Filter,  test  the  precipitate  by  Millon's  test  and 
the  filtrate  by  the  biuret  test.  What  are  your  conclusions? 
(b)  Repeat  the  above  experiment  making  the  saturation  with 
solid  NaCl.  How  does  this  result  differ  from  the  result  of 
the  saturation  with  (NH4)2S04?  Add  2-3  drops  of  acetic 
acid.  What  occurs?  All  proteids  except  peptones  are  preci- 
pitated by  saturating  their  solutions  with  ammonium  sulphate. 
Globulins  are  the  only  proteids  precipitated  by  saturating  with 
XaCl  (see  Globulins,  page  53),  unless  the  saturated  solution  is 
subsequently  acidified,  in  which  event  all  proteids  except  pep- 
tones are  precipitated. 

12.  Coagulation  or  Boiling  Test. — Heat  25  c.c.  of  dilute 
egg  albumin  solution  to  the  boiling-point  in  a  small  evaporating 
dish.  The  albumin  coagulates.  Complete  coagulation  may  be 
obtained  by  acidifying  the  solution  with  3-5  drops  of  acetic 
acid1  at  the  boiling-point.  Test  the  coagulum  by  Millon's 
reaction.  The  acid  is  added  to  neutralize  any  possible  alka- 
linity of  the  solution,  and  to  dissolve  any  substances  which  are 
not  albumin  (see  further  discussion  on  page  292). 

1  Xitric  acid  is  often  used  in  place  of  acetic  acid  in  this  test.     In  case 
nitric  acid  is  used,  ordinarily  1-2  drops  is  sufficient. 

5 


5o 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  19. 


13.  Coagulation  Temperature. — Prepare  4  test-tubes  each 
containing  5  c.c.  of  neutral  egg  albumin  solution.  To  the  first 
add  1  drop  of  0.2  per  cent  HC1,  to  the  second  add  1  drop  of 
0.5  per  cent  Na2COs  solution,  to  the  third  add  1  drop  of  10 
per  cent  NaCl  solution  and  leave  the  fourth  neutral  in  re- 
action. Partly  fill  a  beaker  of  medium  size  with  water  and 
place  it  within  a  second  larger  beaker  which  also  contains 
water,   the  two   vessels  being  separated   by  pieces   of   cork. 

Fasten  the  four  test-tubes  com- 
pactly together  by  means  of  a 
rubber  band,  lower  them  into 
the  water  of  the  inner  beaker 
and  suspend  them,  by  means  of 
a  clamp  attached  to  one  of  the 
tubes,  in  such  a  manner  that 
the  albumin  solutions  shall  be 
midway  between  the  upper  and 
lower  surfaces  of  the  water.  In 
one  of  the  tubes  place  a  ther- 
mometer with  its  bulb  entirely 
beneath  the  surface  of  the  al- 
bumin solution  (Pig.  19) .  Gently 
heat  the  water  in  the  beakers, 
noting  carefully  any  changes 
which  may  occur  in  the  albumin 
solutions  and  record  the  exact 
temperature  at  which  these 
changes  occur.  The  first  appear- 
ance of  an  opacity  in  an  albumin 
solution  indicates  the  commence- 
ment of  coagulation  and  the  tem- 
perature at  which  this  occurs 
should  be  recorded  as  the  coagu- 
lation temperature  for  that  par- 
ticular albumin  solution. 


Coagulation  Temperature 
Apparatus. 


What  is  the  order  in  which  the  four  solutions  coagulate? 


1'ROTEIDS.  5  I 

Repeat  the  experiment  adding  to  the  first  tube  I  drop  of 
acetic  acid,  to  the  second  i  drop  of  concentrated  KOH  solu- 
tion, to  the  third  2  drops  of  a  10  per  cent  NaCl  solution  and 
leave  the  fourth  neutral  as  before. 

What  is  the  order  of  coagulation  here?    Why? 

14.  Precipitation  by  Alcohol. —  Prepart  3  test  tubes  each 
containing  about  ioc.c.  of  95  per  cent  alcohol.  To  the  first  add 
one  drop  of  0.2  per  cent  HC1,  to  the  second  one  drop  of  KOH 
solution  and  leave  the  third  neutral  in  reaction.  Add  to  each 
tube  a  few  drops  of  egg  albumin  solution  and  note  the  result-. 
What  do  you  conclude  from  this  experiment?  Alcohol  pre- 
cipitates proteids  unaltered  but  if  allowed  to  remain  under 
alcohol  the  proteid  is  coagulated.  The  "fixing"  of  tissues 
for  histological  examination  by  means  of  alcohol  is  an  illustra- 
tion of  the  application  of  this  reaction. 

15.  Preparation  of  Powdered  Egg  Albumin. — This  may 
be  prepared  as  follows:  Ordinary  egg-white  finely  divided  by 
means  of  scissors  or  a  beater  is  treated  with  4  volumes  of 
water  and  filtered.  The  filtrate  is  evaporated  on  a  water-hath 
at  about  500  C.  and  the  residue  powdered  in  a  mortar. 

[6.  Tests  on  Powdered  Egg  Albumin. — With  powdered 
albumin  prepared  as  described  above  (by  yourself  or  furnished 
by  the  instructor),  try  the  following  tests: 

(a)  Solubility. 

(b)  Mil  Ion's  Read  ion. 

(c)  Hopkins-Cole  Reaction. — When  used  to  detect  the  pres- 
ence of  proteid  in  solid  form  this  reaction  should  be  conducted 
as  follows:  Place  5  c.c.  of  concentrated  1T2S04  in  a  test-tube 
and  add  carefully,  by  means  of  a  pipette,  3-5  c.c.  of  Hopkins- 
Cole  reagent.  Introduce  a  small  amount  of  the  solid  substance 
to  be  tested,  agitate  the  tube  slightly,  and  note  that  the  sus- 
pended pieces  assume  a  reddish-violet  color,  which  is  the  char- 
acteristic end-reaction  of  the  Hopkins-Cole  test ;  later  the  solu- 
tion will  also  assume  the  reddish-violet  color. 

(d)  Composition  Test. — Heat  some  of  the  powder  in  a  test- 
tube  in  which  is  suspended  a  strip  of  moistened  red  litmus 


52  PHYSIOLOGICAL    CHEMISTRY. 

paper  and  across  the  mouth  of  which  is  placed  a  piece  of  filter 
paper  moistened  with  plumbic  acetate  solution.  As  the  powder 
is  heated  it  chars,  indicating  the  presence  of  carbon;  the  fumes 
of  ammonia  are  evolved,  turning  the  red  litmus  paper  blue  and 
indicating  the  presence  of  nitrogen  and  hydrogen;  the  plumbic 
acetate  paper  is  blackened,  indicating  the  presence  of  sulphur, 
and  the  deposition  of  moisture  on  the  side  of  the  tube  indicates 
the  presence  of  hydrogen. 

( e )  Immerse  a  tube  containing  a  little  powdered  egg  albu- 
min in  boiling  water  for  a  few  moments.  Remove  and  test 
the  solubility  of  the  albumin  according  to  the  directions  given 
under  (a)  above.  It  is  still  soluble.  Why  has  it  not  been 
coagulated?  Repeat  the  above  experiments  with  powdered 
serum  albumin  and  see  how  the  results  compare  with  those 
just  obtained. 

SULPHUR    IN    PROTEID. 

Sulphur  is  believed  to  be  present  in  two  different  forms 
in  the  proteid  molecule.  The  first  form,  which  is  present  in 
greatest  amount,  is  that  loosely  combined  with  carbon  and 
hydrogen.  Sulphur  in  this  form  is  variously  termed  unoxi- 
dized,  loosely  combined,  mcrcaptan  and  lead-blackening  sul- 
phur. The  second  form  is  combined  in  a  more  stable  manner 
with  carbon  and  oxygen  and  is  known  as  oxidized  or  acid 
sulphur. 

Tests  for  Sulphur. 

i.  Test  for  Loosely  Combined  Sulphur. — To  equal  vol- 
umes of  KOH  and  egg  albumin  solutions  in  a  test-tube  add 
1-2  drops  of  plumbic  acetate  solution  and  boil  the  mixture. 
Loosely  combined  sulphur  is  indicated  by  a  darkening  of  the 
solution,  the  color  deepening  into  a  black  if  sufficient  sulphur 
is  present.     Write  the  reactions  for  this  test. 

2.  Test  for  Total  Sulphur  (Loosely  Combined  and  Oxi- 
dized).— Place  the  substance  to  be  examined  (powdered  egg 
albumin)  in  a  small  porcelain  crucible,  add  a  suitable  amount 
of  solid  fusion  mixture  (KOH  and  KN03  mixed  in  the  pro- 


PROTEIDS.  53 

portion  5:1)  and  heat  carefully  until  a  colorless  mixture  re- 
sults. Cool,  dissolve  the  cake  in  a  little  warm  water  and  filter. 
Acidify  the  filtrate  with  11C1,  heat  it  to  the  boiling-point  and 

add  a  small  amount  of  P>aCl._,  solution.  A  white  precipitate 
forms  if  sulphur  is  present.    What  is  this  precipitate? 

GLOBULINS. 

Globulin^  are  simple  proteids  especially  predominant  in  the 
vegetable  kingdom.  They  are  closely  related  to  the  albumins 
and  in  common  with  them  give  all  the  ordinary  proteid  tests. 
Globulins  differ  from  the  albumins  in  being  insoluble  in  water. 
Most  globulins  are  precipitated  from  their  solutions  by  satura- 
tion with  solid  sodium  chloride  or  magnesium  sulphate.  As  a 
class  they  are  much  less  stable  than  the  albumins,  a  fact  shown 
by  the  increasing  difficulty  with  which  a  globulin  dissolves 
during  the  course  of  successive  reprecipitations. 

We  have  used  an  albumin  of  animal  origin  (egg  albumin) 
for  all  the  proteid  tests  thus  far,  whereas  the  globulin  to  be 
studied  will  be  prepared  from  a  vegetable  source.  There 
being  no  essential  difference  between  animal  and  vegetable 
proteids,  the  vegetable  globulin  we  shall  study  may  be  taken 
as  a  true  type  of  all  globulins,  both  animal  and  vegetable. 

Experiments  on  Globulin. 
Preparation  of  the  Globulin. — Extract  20-30  grams  (a 
handful)  of  crushed  hemp  seed  with  a  5  per  cent  solution  of 
XaCl  for  one-half  hour  at  6o°  C.  Filter  while  hot  through  a 
paper  moistened  with  5  per  cent  NaCl  solution  and  allow  the 
filtrate  to  cool  slowly.  The  globulin  is  soluble  in  hot  5  per 
cent  NaCl  solution  and  is  thus  extracted  from  the  hemp  seed, 
but  upon  cooling  this  solution  much  of  the  globulin  separates 
in  crystalline  form.  This  particular  globulin  is  called  edestin. 
It  crystallizes  in  several  different  forms,  chiefly  octahedra 
(see  Fig.  20,  page  54).  (The  crystalline  form  of  excelsin.  a 
proteid  obtained  from  the  Brazil  nut,  is  shown  in  Fig.  2 1 ,  page 
55.  This  vegetable  proteid  crystallizes  in  the  form  of  hex- 
agonal plates.)      Filter  off  the  edestin  and  make  the  follow- 


54 


PHYSIOLOGICAL    CHEMISTRY. 


ing  tests  on  the  crystalline  body  and  on  the  filtrate  which  still 
contains  some  of  the  extracted  globulin. 

Tests  on  Crystallized  Edestin. —  (i)  Microscopical  ex- 
amination (Fig.  20,  below). 

(2)  Solubility. — Try  the  solubility  in  the  ordinary  solvents 
(see  page  4). 

(3)  MiUon's  Reaction. 

(4)  Coagulation  Test. — Place  a  small  amount  of  the  glo- 
bulin in  a  tube,  add  a  little  water  and  boil.  Now  add  dilute 
HC1  and  note  that  the  proteid  no  longer  dissolves.    It  has  been 

coagulated. 

Fig.  20. 


Edestin. 

Tests  on  Edestin  Filtrate. —  (1)  Influence  of  Proteid 
Precipitants. — Try  a  few  proteid  precipitants  such  as  nitric 
acid,  tannic  acid,  picric  acid  and  mercuric  chloride. 

(2)  Biuret  Test. 

(3)  Coagulation  Test. — Boil  some  of  the  filtrate  in  a  test- 
tube.    What  happens  ? 

(4)  Saturation  with  Sodium  Chloride. — Saturate  some  of 
the  filtrate  with  solid  NaCl.  How  does  this  result  differ  from 
that  obtained  upon  saturating  egg  albumin  solution  with  solid 
XaCl? 


PROTEIDS.  55 

(5)  Precipitation  by  Dilution. — Dilute  some  of  the  filtrate 
with  10-15  volumes  of  water.  Why  does  the  globulin  pre- 
cipitate? 

DERIVED  SIMPLE  PROTEIDS. 

These  bodies  are  obtained  from  native  simple  proteids  by 
various  means,  e.  g.,  through  the  action  of  acids,  alkalis,  heat 
or  enzymes,  the  method  of  treatment  to  which  the  native 
proteid  is  subjected  depending  upon  the  particular  class  of 
derived  proteid  desired.  These  modified  bodies  are  classified 
as  albuminates,  proteoses  (or  albumoses),  peptones  and  coagu- 
lated proteids. 

Albuminates. 

The  albuminates  are  derived  proteids  and  are  produced  by 
the  action  of  acids  or  alkalis  upon  the  native  simple  proteids, 
albumins  and  globulins.    There  are  two  classes  of  albuminates, 

Fig.  21. 


EXCELSIN,    THE    PROTEID    OF    THE    BRAZIL    NUT. 

(Drawn  from  crystals  furnished  by  Dr.  Thomas  B.  Osborne,  New 
Haven,  Conn.) 

i.  e.,  acid  albuminate  and  alkali  albuminate.  They  differ  from 
the  native  simple  proteids  principally  in  being  insoluble  in 
NaCl  solution  and  in  not  being  coagulated  except  when  sus- 


56  PHYSIOLOGICAL    CHEMISTRY. 

pended  in  neutral  fluids.  Both  forms  of  albuminate  are  pre- 
cipitated upon  the  neutralization  of  their  solutions.  They  are 
precipitated  by  saturation  with  (NH4)2S04,  and  by  saturation 
with  NaCl  also  if  they  are  dissolved  in  an  acid  solution.  Acid 
albuminate  contains  a  higher  percentage  of  nitrogen  and  sul- 
phur than  the  alkali  albuminate  from  the  same  source  since 
some  of  the  nitrogen  and  sulphur  of  the  original  proteid  is 
liberated  in  the  formation  of  the  latter.  Because  of  this  fact 
it  is  impossible  to  transform  an  alkali  albuminate  into  an  acid 
albuminate,  while  it  is  possible  to  reverse  the  process  and  trans- 
form the  acid  albuminate  into  the  alkali  modification. 

ACID    ALBUMINATE. 

Preparation. — Take  25  grams  of  hashed  lean  beef,  washed 
free  from  the  major  portion  of  blood  and  inorganic  matter, 
and  place  it  in  a  medium-sized  beaker  with  100  c.c.  of  0.2  per 
cent  HC1.  Place  it  on  a  boiling  water-bath  for  one-half  hour, 
filter,  cool  and  divide  the  filtrate  into  two  parts.  Neutralize 
the  first  part  with  dilute  KOH  solution,  filter  off  the  precipi- 
tate of  acid  albuminate  and  make  the  following  tests : 

(1)  Solubility. — Solubility  in  the  ordinary  solvents  (see 
page  4). 

(2)  Milton's  Reaction. 

(3)  Coagulation  Test. — Suspend  a  little  of  the  albuminate 
in  water  (neutral  solution)  and  heat  to  boiling  for  a  few 
moments.  Now  add  1-2  drops  of  KOH  solution  to  the  water 
and  see  if  the  albuminate  is  still  soluble  in  dilute  alkali.  What 
is  the  result  and  why? 

(4)  Test  for  Loosely  Combined  Sulphur  (see  page  52). 

Subject  the  second  part  of  the  solution  to  the  following 
tests : 

(1)  Coagulation  Test. — Heat  some  of  the  solution  to  boil- 
ing in  a  test-tube.    Does  it  coagulate? 

(2)  Biuret  Test. 

(3)  Influence  of  Proteid  Precipitant s. — Try  a  few  proteid 
precipitants  such  as  picric  acid  and  mercuric  chloride.     How 


PROTEIDS.  5  7 

do  the  results  obtained  compare  with  those  from  the  experi- 
ments on  e.y:g'  albumin?      (See  page  47.) 

ALKALI    ALBUMINATE. 

Preparation. — Carefully  separate  the  white-  from  the  yolk 
of  a  hen's  egg  and  place  the  former  in  an  evaporating  dish. 
Add  concentrated  KOH  solution,  drop  by  drop,  stirring  con- 
tinuously. The  mass  gradually  thickens  and  finally  assumes 
the  consistency  of  jelly.  This  is  solid  alkali  albuminate  or 
"  Lieberkiihn's  jelly."  Do  not  add  an  excess  of  KOH  or  the 
jelly  will  dissolve.  Cut  it  into  small  pieces,  place  a  cloth  or 
wire  gauze  over  the  dish  and  by  means  of  running  water  wash 
the  pieces  free  from  adherent  alkali.  Now  add  a  small  amount 
of  water,  which  forms  a  weak  alkaline  solution  with  the  alkali 
within  the  pieces,  and  dissolve  the  jelly  by  gentle  heat.  Cool 
the  solution  and  divide  it  into  two  parts.  Proceed  as  follows 
with  the  first  part:  Neutralize  with  dilute  HC1,  noting  the  odor 
of  the  liberated  H2S  as  the  alkali  albuminate  precipitates. 
Filter  off  the  precipitate  and  test  as  for  acid  albuminate,  page 
56,  noting  particularly  the  sulphur  test.  How  does  this  test 
compare  with  that  given  by  the  acid  albuminate?  Make  tests 
on  the  second  part  of  the  solution  the  same  as  for  acid  albu- 
minate, page  56. 

Proteoses  (or  Albumoses)  and  Peptones. 

Proteoses  are  intermediate  products  in  the  digestion  of  pro- 
teids  by  proteolytic  enzymes,  as  well  as  in  the  decomposition 
of  proteids  by  hydrolysis  and  the  putrefaction  of  proteids 
through  the  action  of  bacteria.  Peptones  are  formed  after 
the  proteoses  and  are  the  last  products  of  the  above  mentioned 
processes  which  still  possess  true  proteid  characteristics.  In 
other  words,  the  proteid  nature  of  the  end-products  of  the 
cleavage  of  the  proteid  molecule  ceases  with  the  peptones,  and 
the  simpler  bodies  formed  from  peptones  are  bodies  of  a  dif- 
ferent type  (see  page  65). 

There  are  several  proteoses  (protoproteose,  heteroproteose 
and  deuteroproteose),  and  at  least  two  peptones  (amphopep- 


58  PHYSIOLOGICAL    CHEMISTRY. 

tone  and  antipeptone),  which  result  from  proteolysis.  The 
differentiation  of  the  various  proteoses  and  peptones  at  present 
in  use  is  rather  unsatisfactory.  These  compounds  are  classi- 
fied according  to  their  varying  solubilities,  especially  in 
(NH4)2S04  solutions  of  different  strengths.  The  exact  dif- 
ferences in  composition  between  the  various  members  of  the 
group  remains  to  be  more  accurately  established.  Because  of 
the  difficulty  attending  the  separation  of, these  bodies,  pure 
proteose  and  peptone  are  not  easy  to  procure.  The  so-called 
peptones  sold  commercially  contain  a  large  amount  of  proteose. 
As  a  class  the  proteoses  and  peptones  are  very  soluble,  dif- 
fusible bodies  which  are  non-coagulable  by  heat.  Peptones 
differ  from  proteoses  in  being  more  diffusible,  non-precipitable 
by  (NH4)2S04,  and  by  their  failure  to  give  any  reaction  zvith 
potassium  ferrocyanide  and  acetic  acid,  potassio-mer  curie 
iodide  and  HC1,  picric  acid,  and  trichloracetic  acid.  The  so- 
called  primary  proteoses  are  precipitated  by  HN03  and  are 
the  only  members  of  the  proteose-peptone  group  which  are 
so  precipitated. 

Some  of  the  more  general  characteristics  of  the  proteose- 
peptone  group  may  be  noted  by  making  the  following  simple 
tests  on  a  proteose-peptone  powder : 

(1)  Solubility. — Solubility  in  the  ordinary  solvents  (see 
page  4). 

(2)  Milton's  Reaction. 

Dissolve  a  little  of  the  powder  in  water  and  test  the  solu- 
tion as  follows : 

(1)  Precipitation  by  Picric  Acid. — To  5  c.c.  of  proteose- 
peptone  solution  in  a  test-tube  add  picric  acid  until  a  perma- 
nent precipitate  forms.  The  precipitate  disappears  on  heating 
and  returns  on  cooling. 

(2)  Precipitation  by  a  Mineral  Acid. — Try  the  precipita- 
tion by  nitric  acid. 

(3)  Coagulation  Test. — Heat  a  little  proteose-peptone  solu- 
tion to  boiling.  Does  it  coagulate  like  the  other  simple  pro- 
teids  studied? 


PROTEIDS.  59 

SEPARATION  OF  PROTEOSES  AND  PEPTONES. 

Place  50  c.c.  of  proteose-peptone  solution  in  an  evaporating 
dish  or  casserole,  and  half-saturate  it  with  (NH4)2S04  solu- 
tion, which  may  be  accomplished  by  adding  an  equal  volume 
of  saturated  (NH4)2S04  solution.  At  this  point  note  the 
appearance  of  a  precipitate  of  the  primary  proteoses  (proto- 
proteose  and-heteroproteose).  Now  heat  the  half -saturated 
solution  and  its  suspended  precipitate  to  boiling  and  saturate 
the  solution  with  solid  (NH4).,S04.  At  full  saturation  the 
secondary  proteoses  (deuteroproteoses)  are  precipitated.  The 
peptones  remain  in  solution. 

Proceed  as  follows  with  the  precipitate  of  proteoses :  Col- 
lect the  sticky  precipitate  on  a  rubber-tipped  stirring  rod  or 
remove  it  by  means  of  a  watch  glass  to  a  small  evaporating 
dish  and  dissolve  it  in  a  little  water.  To  remove  the 
(NH4)2S04,  which  adhered  to  the  precipitate  and  is  now  in 
solution,  add  BaCOa,  boil,  and  filter  off  the  precipitate  of 
BaS04.  Concentrate  the  proteose  solution  to  a  small  volume1 
and  make  the  following  tests : 

( 1 )  Biuret  Test. 

(2)  Precipitation  by  HN03. — What  would  a  precipitate 
at  this  point  indicate? 

(3)  Precipitation  by  Trichloracetic  Acid. — This  precipitate 
dissolves  on  heating  and  returns  on  cooling. 

(4)  Precipitation  by  Picric  Acid. — This  precipitate  also 
disappears  on  heating  and  returns  on  cooling. 

(5)  Precipitation  by  Potassio-mercuric  Iodide  and  HC1. 

(6)  Coagulation  Test. — Boil  a  little  in  a  test-tube.  Does 
it  coagulate? 

(7)  Acetic  Acid  and  Potassium  Ferrocyanide  Test. 

The  solution  containing  the  peptones  should  be  cooled  and 

*If  the  proteoses  are  desired  in  powder  form,  this  concentrated  proteose 
solution  may  now  be  precipitated  by  alcohol,  and  this  precipitate,  after 
being  washed  with  absolute  alcohol  and  with  ether,  may  be  dried  and 
powdered. 


60  PHYSIOLOGICAL    CHEMISTRY. 

filtered,  and  the  (NH4)2S04  in  solution  removed  by  boiling 
with  BaC03  as  described  on  page  59.  After  filtering  off  the 
BaS04  precipitate,  concentrate  the  peptone  filtrate  to  a  small 
volume1  and  repeat  the  tests  as  given  under  the  proteose  solu- 
tion, page  59.  In  the  biuret  test  the  solution  should  be  made 
very  strongly  alkaline  with  solid  KOH. 

Coagulated  Proteids. 

These  derived  proteids  are  produced  from  unaltered  proteid 
materials  by  heat,  by  long  standing  under  alcohol,  or  by  the 
continuous  movement  of  their  solutions  such  as  that  produced 
by  rapid  stirring  or  shaking.  In  particular  instances,  such  as 
the  formation  of  fibrin  from  fibrinogen  (see  page  157),  the 
coagulation  may  be  produced  by  ferment  action.  Ordinary 
soluble  proteids  after  having  been  transformed  into  the  coag- 
ulated modification  are  no  longer  soluble  in  the  ordinary  sol- 
vents. Upon  being  heated  in  the  presence  of  strong  acids  or 
alkalis,  coagulated  proteids  are  converted  into  albuminates. 

Many  proteids  coagulate  at  an  approximately  fixed  tem- 
perature under  definite  conditions  (see  page  50).  This  char- 
acteristic may  be  applied  to  separate  different  coagulable  pro- 
teids from  the  same  solution  by  fractional  coagulation.  The 
coagulation  temperature  frequently  may  serve  in  a  measure 
to  identify  proteids  in  a  manner  similar  to  the  melting-point 
or  boiling-point  of  many  other  organic  substances.  The 
separation  of  proteids  by  fractional  coagulation  thus  is 
analogous  to  the  separation  of  volatile  substances  by  means 
of  fractional  distillation.  The  nature  of  the  process  in- 
volved in  the  coagulation  of  proteids  by  heat  is  not  well 
understood,  but  it  is  probable  that  in  addition  to  the  altered 
arrangement  of  the  component  atoms  in  the  molecule,  there 
is  a  mild  hydrolysis  which  is  accompanied  by  the  liberation  of 
minute  amounts  of  hydrogen,  nitrogen  and  sulphur.  The 
presence  of  a  neutral  salt  or  a  trace  of  a  mineral  acid  may 
facilitate  the  coagulation  of  a  proteid  solution  (see  page  50), 

1  See  note  on  preparation  of  proteose  powder,  page  59. 


PROTEIDS.  6 1 

whereas  any  appreciable  amount  of  acid  or  alkali  will  retard 
or  entirely  prevent  such  coagulation. 

Experiments  on  Coagulated  Proteid. 

Ordinary  coagulated  egg-white  may  be  used  in  the  following 

tests : 

i.  Solubility. — Try  the  solubility  of  small  pieces  of  the 
coagulated  proteid  in  each  of  the  ordinary  solvents  (see 
page  4). 

_'.   Millon's  Reaction. 

3.  Xanthoproteic  Reaction. — Partly  dissolve  a  medium- 
sized  piece  of  the  proteid  in  concentrated  HXO.;.  Cool  the 
solution  and  add  an  excess  of  NH4OH.  Both  the  proteid 
solution  and  the  undissolved  proteid  will  be  colored  orange. 

4.  Biuret  Test. — Partly  dissolve  a  medium-sized  piece  of 
the  proteid  in  concentrated  KOH  solution.  If  the  proper  dilu- 
tion of  CuS04  solution  is  now  added  the  white  coagulated 
proteid,  as  well  as  the  proteid  solution,  will  assume  the  char- 
acteristic purplish-violet  color. 

5.  Hopkins-Cole  Reaction. — Conduct  this  test  according 
to  the  modification  given  on  page  51. 


COMPOUND  PROTEIDS. 

Compound  proteids  consist  of  a  simple  proteid  combined 
with  some  non-proteid  material,  and  they  are  named  accord- 
ing to  the  nature  of  this  combining  body.  Thus  we  have 
glucoprotcids.  nucleo proteids  and  Jiccmoglobins  as  three  classes 
of  compound  proteids. 

The  glucoprotcids  yield,  upon  decomposition,  proteid  and 
carbohydrate  derivatives,  notably  glucosamine,  CH2OH  ■  - 
(CHOH)3  •  CH(NH2)  •  CHO,  and  galactosamine,  OHCH2  ■  - 
(CHOH)3-CH(NH2)-CHO.  The  principal  glucoproteids 
are  mucoids,  mucins  and  chondroprotcids.  By  the  term 
mucoid  we  may  designate  those  glucoproteids  which  occur  in 
tissues,    such    as    tendomucoid    from    tendinous    tissue    and 


62  PHYSIOLOGICAL    CHEMISTRY. 

osseomucoid  from  bone.    The  elementary  composition  of  these 
typical  mucoids  is  as  follows : 


N. 

s. 

C. 

H. 

0. 

Tendomucoid 

..11.75 

2-33 

48.76 

6-53 

30.60  (Chittenden  and  Gies) 

Osseomucoid    . 

.  12.22 

2.32 

47-43 

6.63 

31.40 

The  term  mucins  may  be  said  to  include  those  forms  of 
glucoproteids  which  occur  in  the  secretions  and  fluids  of  the 
body.  Chondroproteids  are  so  named  because  chondromncoid, 
the  principal  member  of  the  group,  is  derived  from  cartilage 
(chondrigen).  Amyloid,  which  appears  pathologically  in  the 
spleen,  liver  and  kidneys  is  also  a  chondroproteid. 

The  nucleoproteids  occur  principally  in  animal  and  vegetable 
cells,  and  following  the  destruction  of  these  cells  they  are 
found  in  the  fluids  of  the  body.  These  proteids  are  discharged 
into  the  tissue  fluids  by  the  activity  or  disintegration  of  cells. 
Combined  with  the  simple  proteid  in  the  nucleoproteid  mole- 
cule we  find  nucleic  acid,  a  body  which  contains  phosphorus 
and  which  yields  pitrin  bases  upon  decomposition.  The  so- 
called  nucleins  are  formed  in  the  gastric  digestion  of  nucleo- 
proteids. 

The  hemoglobins  are  those  compound  proteids  which  are 
composed  of  a  simple  proteid  and  a  pigment.  The  haemoglobin 
of  the  blood  (see  page  156)  upon  decomposition  yields  a  pro- 
teid termed  globin  and  a  modified  pigment  called  hccmatin. 

For  experiments  upon  a  compound  proteid  see  page  199. 

ALBUMINOIDS,  ALBUMOIDS  OR  PROTEOIDS. 

These  bodies  are  closely  related  in  character  to  the  proteids, 
from  which  class  of  substances  they  are  derived.  They  differ 
ordinarily  from  true  proteids  in  the  character  of  their  decom- 
position products,  in  being  very  resistant  to  the  ordinary  pro- 
teid solvents,  and  in  being  unable  alone  to  support  life.  They 
generally  occur  in  an  insoluble  form  in  some  portion  of  the 
animal  organism.  The  albuminoids  may  be  divided  into  sev- 
eral classes  such  as  keratins,  elastins,  collagcns,  gelatins  and 


I'KOTKIDS. 


63 


skeletons,  and  in  general  the  members  of  each  group  differ 
fundamentally  in  certain  characteristics  from  the  member-  of 
any  other  group.  For  discussion  of  and  experiments  on  each 
of  the  several  groups  see  the  chapter  on  Epithelial  and  Con- 
nective  Tissues,  pages  \ny  to  205. 


REVIEW  OF  PROTEIDS. 

In  order  to  facilitate  the  student's  review  of  the  proteids, 
the  preparation  of  a  chart  similar  to  the  appended  model  is 
recommended.  The  signs  -f-  and  —  may  be  conveniently 
used  to  indicate  positive  and  negative  reactions. 


MODEL   CHART 

FOR    REVIEW    PURPOSES. 

Troteid. 

Solubility. 

8 
H 
u 

0 

"o 
U 

!2 
'5 

0 
u 
ft. 

Precipitation  Tests. 

SaltinR- 

..111 
Tests. 

1     5 

~    -5 
x     ' 

z 

a 

0 

1 

a 

- 

X 

>■ 

ja 

a 
.0 

a 

1 
0 
U 

V 

s 

u 

Z 

X*. 

0 

u 

x 

6 

0" 
U 

Z 
•*. 

m 
d 

D 

x 

6 
c 
0 
■J 

tn 

O 
ui 

c 
0 
U 

!2 

IS 

3 

s 

"0 

X 

0 
u 

5 

V 

■a    . 
"52 

O_o 

1+ 

3^ 

E  + 
0  u 

3,2 

04 

•6 

< 
0 

0 

V 

0 
H 

Albumin. 

Globulin. 

Acid  albuminate. 

Alkali  albuminate. 

Proteose. 

Peptone. 

Coagulated  proteid. 

"  Unknown  "  Mixtures  and  Solutions  of  Proteids. 

At  this  point  the  student's  knowledge  of  the  characteristics 
of  the  various  proteids  studied  will  be  tested  by  requiring  him 
to  examine  several  "  unknown  "  proteid  mixtures  or  solutions 
and  make  full  report  upon  the  same.  The  scheme  given  on 
page  64  may  be  used  in  this  examination. 


64 


PHYSIOLOGICAL    CHEMISTRY. 


oti 

u 


S. 


w 

H 

O 
p4 

Oh 

o 
o 

U 
W 
H 

W 

O 

w 

H 


O 

a 

2 

w 

U 


a  «<i 

e  c 

e  B 

'3  » 

.2  «i 


T3   C   u 

G  V 

rt  s- 

Il- 
ia,; 

o  g  3 


§2 


3.   *§•- 


"5  .  tj  § 
b  >>22 

"3—   JJ   3~ 

Z  wiE  g 


«  a" 

V     yj     U 

Jgjj 

-°    o   M 

2  =  « 

O    tj    U  - 
«-.    ~    11 

at  c  5 

=  '=-€ 

—  3  * 

rt     ~     ^ 

^■5.5 
*  h  a 

-  =  a 


5  j! 


2  c  _ 


ote"  "•> 
■-  o  o 


3s 

2j= 


2~ 
■Eg 

Id 

.2*  3 
'S  ft 


i  -^  >.. 


§£ 


CO  o 

-a  o 
.So. 

3  D-S 
-".§-§  « - 


>£>  ■» 


3  §-9 

o.«    d  be 
•3  fc  =§ 

^  S-C.t! 
u       ~  ft 

-s  ss « 

_"o*22 

ffl  a  rt  q 


«  5j  s; 

p    ft    ^   t) 


•*!    3 

1>4) 


5* 

4!  e 


'■a  & 

■a  o 

"■V    c 


gsuS 

•o-S.*'  " 

—  u  '    — 
6  o      o 

beg  o-~ 
o-o       o 

Soft 

5  u  g  " 

a  rt  us 

O    «,^   u 

|||  g 


g  .S  ^  g  -a  - 

£  J3    q   3    s  u 
1)2    ^   O    >•  < 

j3  he  a  <"CQ 

-2-o  e  5" 
«  c  ?  S   • 

S  S*8i° 

-«■    °£ 

v  rt  S  o>Ea 

E?  o  v  % 
tflJ3    U.2 


(o 


.•«&< 


BO  < 

SCfl-; 

o  ^.: 

i— .is  i 

'15  S  i 

3ft   i 


2.St3? 


&)  o 

rt  ft 

0)  '       *J 
'ft^   o 


!-§  s 

3  'v*   •« 

-oe<3  -J< 
g«    §" 

U2  K 

U      0) 


S-^S225  g  a 

^  -^  O        *j  -^        -^ 
— ..«    X  -   o   o   „,   . 

^7  «c^   ■§.«  m 

?Jc  «  M-  -G  2  g 
•^2'"„-  «  *  s  2  « 
A1-^  u'O.Su  fe-S  be 

^    ft—  n    n  *-   l_u  ft  IT 

S-Sag 


ft  tfl    O   ft  cj 


■*->  j2  •,-;  rt  S 
5  2  2  «  -2  « 
.g  rt  ov.  P  h 

—  a .-  ~  f  2  "5 
^  " >>  S  rt  u 

<u2 


•S.I 


x2 


c 

a 

03 

a 

_rt 

S 

_rt 

"a; 

he  cis 

a 

0 

<U  T3 

0) 

c 

g 

■d 

^ 

2 

0 

2 

? 

«  u       -«  g 

:*••£  ^3  v  o. 
^^  5  ctl2? 


^   3   u   -   «u 

S2  s  §r 


■5  « 


CHAPTER    IV. 

DECOMPOSITION    PRODUCTS    OF 
PROTEIDS. 

Although  various  physico-chemical  considerations  indicate 
that  the  proteid  molecule  is  very  large,  approaching  a  molecular 
weight  of  15.000  (T.  B.  Osborne),  more  definite  statements 
on  this  point  cannot  be  made  at  the  present  time;  nor  can  any 
definite  constitutional  formula  yet  be  assigned  to  any  of  the 
proteid  substances.  Notwithstanding  this,  by  a  study  of  the 
decomposition  products  of  proteids  much  has  been  learned  re- 
garding the  inner  structure  of  the  complex  molecule;  our 
knowledge  in  this  direction  has  recently  been  greatly  advanced. 
Decomposition  may  be  brought  about  by  oxidation  or  by  hy- 
drolysis, the  end-products  of  the  decomposition  varying  more 
01  less  in  character,  according  to  the  nature  of  the  process. 
Oxidation  is  ordinarily  facilitated  by  the  use  of  such  oxidizing 
agents  as  potassium  permanganate,  hydrogen  peroxide  or  bro- 
mine, while  hydrolysis  is  brought  about  by  acids,  alkalis  or 
superheated  steam,  and  in  digestion  by  the  action  of  the  pro- 
teolytic enzymes.  Among  the  decomposition  products  of  pro- 
teids are  proteoses,  peptones,  carbon  dioxide,  ammonia,  hydro- 
gen sulphide,  amines,1  amides,2  tryptophan,  mono-amino  acids 
(such  as  leucin,  tyrosin,  cysiin,  aspartic  acid,  glutamic 
acid,  glycocoll,  alanin,  phenylalanin ,  amino-valerianic  acid, 
prolin,  oxyprolin,  serin)  and  di-amino  acids,  such  as  arginin. 
lysin  and  liistidiu. 

Proteids  may  also  be  decomposed  by  putrefactive  bacteria 
with  the  formation  of  such  bodies  as  phenol,  para-cresol,  indol, 
skatol,  etc.  (see  page  129). 

1  An  amine  is  a  body  formed  after  the  type  of  ammonia,  one  or  more 
hydrogen  atoms  being  replaced  by  hydrocarbon  radicals. 

2  An  amide  is  a  body  formed  after  the  type  of  ammonia,  one  or  more 
hydrogen  atoms  being  replaced  by  organic  acid  radicals,  i.  e.,  that  portion 
of  the  acid  left  after  removing  the  hydroxyl  group. 

6  65 


66  PHYSIOLOGICAL    CHEMISTRY. 

For  the  benefit  of  those  especially  interested  in  such  matters 
a  photograph  of  the  Fischer  apparatus  (Fig.  22,  page  67)  used 
in  the  fractional  distillation,  in  vacuo,  of  the  esters  of  the 
decomposition  products  of  the  proteids,  as  well  as  micro- 
photographs  and  drawings  of  preparations  of  several  of  these 
decomposition  products  (Figs.  —  to  — ,  pp.  —  to  — )  are 
introduced.  For  the  preparations  and  the  photograph  of  the 
apparatus  the  author  is  indebted  to  Dr.  T.  B.  Osborne,  of 
New  Haven,  Conn.  These  drawings  and  photographs  are 
not  introduced  at  this  point  through  any  preconceived  notion 
that  the  student  will  derive  any  practical  benefit  therefrom, 
but  are  rather  inserted  with  the  idea  of  giving  him  a  graph- 
ical illustration  of  the  magnitude  of  the  proteid  molecule, 
and  with  the  hope  that  they  may  perhaps  act  as  a  stimulus, 
which  will  cause  him  to  desire  a  more  extended  knowledge  of 
the  science  of  physiological  chemistry,  and,  in  particular,  of 
that  important  class  of  substances,  the  proteids.  The  repro- 
duction of  the  crystalline  form  of  some  of  the  more  recent  of 
the  products  may  be  of  interest  to  those  viewing  the  field  of 
physiological  chemistry  from  other  than  the  student's  aspect. 

Any  extended  discussion  of  the  various  decomposition  prod- 
ucts being  out  of  place  in  a  book  of  this  character,  we  will 
simply  make  a  few  general  statements  in  connection  with  the 
more  important  products. 

DISCUSSION    OF    THE    PRODUCTS. 

Tyrosin,  CgH^NOs. — Tyrosin,  one  of  the  most  important 

end-products    of   proteid   decomposition,    is   the   amino   acid, 

p-oxyphcnyl-a-amino-propionic    acid.      It    has    the    following 

formula : 

NH2 

I 
CHo-CH-COOH. 


OH 


DECOMPOSITION     PRODUCTS    OF    PROTEIDS. 


67 


Tyrosin  occurs  in  conspicuous  amounts  as  an  end-product  of 
the  pancreatic  digestion  of  proteids  (see  page  107),  and  is 
generally  accompanied  by  leucin.     It  does  not  occur,  however, 


Fig.  22. 


Fischer  Apparatus. 

Reproduced  from  a  photograph  made  by  Prof.  E.  T.  Reichert,  of  the 
University  of  Pennsylvania.  The  negative  was  furnished  by  Dr.  T.  B. 
Osborne,  of  New  Haven,  Conn. 

./.  Tank  into  which  freezing  mixture  is  pumped  and  from  which  it  flows 
through  the  condenser,  B  ;  C,  flask  from  which  the  esters  are  distilled,  the  dis- 
tillate being  collected  in  D  ;  E,  a  Dewar  flask  containing  liquid  air  serving 
as  a  cooler  for  condensing  tube  F ;  G  and  G' ,  tubes  leading  to  the  Geryck 
pump  by  which  the  vacuum  is  maintained  :  /,  tube  leading  to  a  McLeod  guage 
(not  shown  in  figure)  ;  J ,  a  bath  containing  freezing  mixture  in  which  the 
receiver  D  is  immersed  ;  K,  a  bath  of  water  during  the  first  part  of  the  dis- 
tillation and  of  oil  during  the  last  part  of  the  process  ;  1-5.  stop  cocks  which 
permit  the  cutting  out  of  different  parts  of  the  apparatus  as  the  procedure 
demands. 


68 


PHYSIOLOGICAL    CHEMISTRY. 


as  an  end-product  of  the  decomposition  of  gelatin.  Tyrosin 
and  leucin  have  the  power  of  forming  salts  with  copper.  Since 
the  tyrosin  salt  is  much  less  soluble  than  the  leucin  salt,  this 
forms  the  basis  for  a  method  of  separating  these  bodies. 

Fig.  23. 


Tyrosin. 


Tyrosin  is  found  in  old  cheese,  and  derives  its  name  from 
this  fact.  It  crystallizes  in  tufts,  sheaves  or  balls  of  fine 
needles,  which  melt  at  295  °  C.  and  are  rather  insoluble  in 
cold  (1-2454)  or  boiling  (1-154)  water.  It  is  soluble  in 
alkalis,  ammonia  or  mineral  acids,  and  less  easily  soluble  in 
acetic  acid  or  hot  95  per  cent  alcohol.  Tyrosin  responds  to 
Millon's  reaction,  thus  showing  the  presence  of  the  hydroxy- 
phenyl  group,  but  gives  no  other  proteid  test.  In  severe  cases 
of  typhoid  fever  and  smallpox,  in  acute  yellow  atrophy  of  the 
liver,  and  in  acute  phosphorus  poisoning,  tyrosin  has  been 
found  in  the  urine.  Tyrosin  crystals  obtained  as  a  decom- 
position product  of  the  proteid  gliadin  are  shown  in  Fig.  23, 
above. 

Leucin,  C6H13N02. — Leucin  is  an  important  end-product 
of  the  decomposition  of  proteid  material,  and  was  the  first  of 
these  products  to  be  discovered  (1818).     It  is  an  amino  acid, 


DECOMPOSITION     PRODUCTS    OF    l'KOTF.IDS. 


69 


a-atnino-isobutyl-acetic  acid,  and  therefore  lias  the  following 
formula  : 

CFI,  NH2 

I  L 

CH-CH,-C-COOII. 


(II. 


H 


1 1  is  present  normdlly  in  the  pancreas,  thymus,  thyroid,  spleen, 
brain,  liver,  kidneys  and  salivary  glands.  It  has  been  found 
pathologically  in  the  urine  (in  acute  yellow  atrophy  of  the  liver, 
in  acute  phosphorus  poisoning  and  in  severe  cases  of  typhoid 
fever  and  smallpox),  and  in  the  liver,  blood  and  pus. 

Fig.  24. 


Leucin, 

Pure  leucin  crystallizes  in  thin,  white  hexagonal  plates. 
Crystals  of  pure  leucin,  obtained  as  a  decomposition  product  of 
the  proteid  gliadin,  are  reproduced  in  Fig.  24.  Impure  leucin 
is  a  slightly  refractive  substance,  which  generally  crystal- 
lizes in  balls  having  a  radial  structure  or  in  aggregations  of 
spherical  bodies.  Fig.  104,  page  326.  It  is  rather  easily  soluble 
in  water  (46  parts),  alcohol,  alkalis,  ammonia  and  acids.  On 
heating  to  1700  C,  leucin  sublimes  with  the  formation  of  car- 
bon dioxide,  ammonia  and  amylamine.  In  aqueous  solutions 
leucin  is  laevorotatory,  whereas  in  acid  or  alkaline  solutions  it 
is  dextrorotatory. 


70 


PHYSIOLOGICAL    CHEMISTRY 


Aspartic   Acid,    C4H7N04. — Chemically,    aspartic   acid   is 
amino-succinic  acid  and  has  the  following  structural  formula: 

XH, 

I 
CH-COOH 

I 
CH2-COOH. 

The  amide  of  aspartic  acid,  asparagin,  is  very  "widely  dis- 
tributed in  the  vegetable  kingdom.  The  crystalline  form  of 
aspartic  acid.,  as  obtained  from  the  proteid  gluienin,  is  exhibited 

in  Fig.  25. 

Fig.  25. 


Aspaktic  Acid. 

Glutamic  Acid,  C-;H0XO4. — This  acid  is  a-amino-normal- 
glutaric  acid  and  as  such  bears  the  following  graphic  formula: 

XH. 

I 
CH-COOH 

I 
CHo 

I 
CH..-COOH. 


DECOMPOSITION     PRODUCTS    OF    I'KOTF.IDS. 


71 


Upon  hydrolyzing  the  wheat  proteid  gliadin  with  hydrochloric 
acid,  Osborne  and  Harris  obtained  a  yield  of  37  per  cent 
of  glutamic  acid.     This  is  the  largesl  amount  of  an  amino 


Fig.  26. 


Glutamic  Acid. 

Reproduced   from  a  micro-photograph   made  by   Prof.   E.   T.   Reichert,   of  the 

University   of    Pennsylvania. 

acid  yet  obtained  as  a  decomposition  product  of  any  proteid 
substance.  Crystals  of  glutamic  acid,  obtained  from  the  pro- 
teid gliadin,  are  reproduced  in  Fig.  26. 

Glycocoll,  C2H5N02. — Glycocoll,  or  amino  acetic  acid,  is 
the  simplest  of  the  amino  acids  and  has  the  following 
formula : 

NH2 

H-C-COOH. 

H 

It  was  the  second  of  the  decomposition  products  of  proteids  to 
be  discovered,  being  preceded  only  by  leucin.  In  distinction 
from  the  greater  number  of  the  amino  acids,  glycocoll  may  be 
determined  quantitatively  very  accurately.  Hippuric  acid  may 
be  formed  synthetically  through  a  union  of  glycocoll  and  ben- 


72 


PHYSIOLOGICAL    CHEMISTRY, 


zoic  acid  (see  page  263).  This  method  is  not  used  very  exten- 
sively at  present,  however,  having  been  replaced  by  Fischer's 
method.  Glycocoll,  ingested  in  small  amount,  is  excreted  in 
the  urine  as  urea,  whereas  if  administered  in  excess  it  appears 
in  part  unchanged  in  the  urine.  The  crystalline  form  of  gly- 
cocoll ester  hydrochloride,  resulting  from  the  decomposition 
of  the  proteid  glutenin,  is  shown  in  Fig.  27. 

Fig.  2j. 


Glycocoll  Ester  Hydrochloride. 

Alanin,  C3H7N02. — From  a  chemical  view-point  this  de- 
composition product  is  a-amino-propionic  acid,  and  as  such  it 
may  be  represented  structurally  as  follows : 

H    NH, 


H-C-C-COOH. 

I       I 
H     H 

Obtained  from  proteid  substances,  alanin  is  dextrorotatory,  is 
very  easily  soluble  in  water,  and  possesses  a  sweet  taste. 
Tyros-in,  phenylalanin,  cystin  and  serin  are  derivatives  of 
alanin. 


DFXOMPOSITION    PRODUCTS    OF    PROTEIDS. 


73 


Phenylalanin,     C9HnN02. — This     product     is     phenyl  a 

amino-propionic  acid,  and  may  be  represented  graphically  as 

follows : 

H    NH2 

I      I 
(<     C-COOH. 

• .  Un  H 

Phenylalanin  is  not  so  soluble  as  alanin,  and  possesses  a  sweet 
taste.  The  yield  of  this  body  from  the  decomposition  of  pro- 
teids  is  frequently  greater  than  the  yield  of  tyrosin.  The 
crystalline  form  of  phenylalanin  obtained  from  the  proteid 
gliadin  is  shown  in  Fig.  28. 

Fig.  28. 


Phenylalanin. 


Amino-valerianic  Acid,  C5HnN02. — This  acid  is  prob- 
ably a-amino-isoralcrianic  acid,  and  as  such  bears  the  follow- 
ing formula : 

CH3  NH2 


H-C— C-COOH. 

I         I 
CH3  H 


74 


PHYSIOLOGICAL    CHEMISTRY. 


It  closely  resembles  leucin  in  many  of  its  properties,  and  for 
this  reason  is  difficult  to  identify  in  the  presence  of  leucin.  It 
is  quite  readily  soluble  in  water  and  is  dextrorotatory. 

Prolin,  C5H9N02. — Chemically,  prolin  is  a-pyrrolidin- 
carboxylic  acid  and  therefore  possesses  the  following  graphic 
structure : 

H2C CH2 


H,C\/CH-COOH. 
NH 

Prolin  was  first  obtained  as  a  decomposition  product  of  casein, 
is  lsevorotatory  and  possesses  a  very  sweet  taste.  The  crystal- 
line form  of  lecvo-a-prolin  is  shown  in  Fig.  29,  and  the  copper 

Fig.  pg. 


LjEVO-<x-Prolin. 


salt  of  prolin  is  represented  by  a  micro-photograph  in  Fig. 
30,  page  75.  Both  were  obtained  from  the  proteid  gliadin. 
The  crystals  of  the  copper  salt  possess  a  faint  bluish  tinge. 

Serin,  C3H7N03. — Serin,  from  a  chemical  standpoint,  is 
a-aniino-fS-hydroxy-propionic  acid  and  possesses  the  follow- 
ing structural  formula : 


DECOMPOSITION-     PRODUCTS    OF    PROTEIDS. 

OH  XII. 

I        I 
H-  C      i1  — COOK. 

I        I 
H      II 

Fig.  30. 


75 


Copper  Salt  of  Pkoi.ix. 

Reproduced   from   a   micro-photograph   made  by   Prof.   E.   T.   Reichert,   of   the 

University  of   Pennsylvania. 

Fig.  31. 


Si: ''IN. 


76 


PHYSIOLOGICAL    CHEMISTRY. 


This  product  is  inactive,  possesses  a  sweet  taste  and  is  quite 
readily  soluble  in  hot  water.  Serin  crystals,  obtained  as  a 
decomposition  product  of  the  proteid  gliadin,  are  shown  in  Fig. 
31,  page  75. 

Cystin,    C0Ht2O4N2S2. — Friedmann    has    recently    shown 
cystin  to  possess  the  following"  structural  formula : 


CH2  •  S— — S  •  CH2 


CH-NIL 

I 
COOH 


CH- 


NH< 


COOH. 


Cystin  is  the  principal  sulphur-containing  body  obtained 
from  the  decomposition  of  proteid  substances.  It  is  obtained 
in  greatest  amount  as  a  decomposition  product  of  such  keratin- 
containing  tissues  as  horn,  hoof  and  hair.     Cystin  occurs  in 

Fig.  32. 


Cystin. 

small  amount  in  normal  urine  and  is  greatly  increased  in 
quantity  under  certain  pathological  conditions.  It  crystal- 
lizes in  the  form  of  hexagonal  plates  which  are  thin  and  color- 
less; crystals  obtained  from  the  decomposition  of  the  proteid 
gliadin  are  shown  in  Fig.  32.     Cystin  is  soluble  in  alkalis, 


DECOMPOSITION    PRODUCTS    OF    PROTEIDS.  77 

ammonia,  oxalic  acid  solution  and  mineral  acids  but  practically 
insoluble  in  water,  acetic  acid,  alcohol  and  ether.  It  is 
laevorotatory. 

It  has  recently  been  claimed  that  cystin  occurs  in  two  forms, 
i.  e.,  stone-cystin  and  proteid-cystin  and  that  these  two  forms 
are  distinct  in  their  properties.     This  view  Is  incorrect. 

For  a  discussion  of  cystin  sediments  in  urine  see  Chapter 
XIX. 

Tryptophan,  C^H^N-jOo. — According  to  Ellinger,  trypto- 
phan is  indol-amino-propionic  acid.  Until  very  recently  this 
investigator  thought  the  following  was  the  correct  structural 
formula  of  this  substance  : 

NH2 

I 
H-C-H 

I 
C-C-COOH. 

I 

H 
\/\/CH 
NH 

Further  investigation  by  him,  however,  has  shown  this  view 
to  be  incorrect.  He  now  proposes  the  two  formulas  which 
follow  and  expects  further  study  will  show  definitely  which 
one  correctly  represents  the  structure  of  tryptophan : 

/\ C-CHo-CH(NH«,)-COOH 

I      J      II 

NH 


or 


/\ C-CH(NH2)-CH2-COOH. 


u 


\/CH 
NH 

Tryptophan  is  the  mother-substance  of  indol  and  its  presence 
in  proteid  substances  may  be  shown  by  means  of  the  Adam- 


78  PHYSIOLOGICAL    CHEMISTRY. 

kiewicz  reaction  or  the  Hopkins-Cole  reaction  (see  p.  45).  It 
may  be  detected  in  a  pancreatic  digestion  mixture  through  its 
property  of  giving  a  violet  color-reaction  with  bromine  water. 
Lysin,  C0H14N2O2. — The  three  bodies,  lysin,  arginin  and 
histidin,  are  frequently  classed  together  as  the  hexone  bases. 
Lysin  was  the  first  of  the  bases  discovered.  It  is  a-e-diamino- 
caproic  acid  and  hence  possesses  the  following  structure: 

NH,  H    H    H    NHo 

I        I      I      I      I 
H-C  —  C-C-C-C-COOH. 

H     H    H    H    H 

It  is  dextrorotatory  and  is  found  in  largest  amount  in  casein 
and  gelatin.     It  is  the  mother-substance  of  cadaverin  and  has 

Fig.  33. 


Lysin  Picrate. 

never  been  obtained  in  crystalline  form.  The  picrate  of  lysin 
may  be  crystallized,  however;  the  crystals  of  this  body,  ob- 
tained from  the  proteid  legumin,  are  shown  in  Fig.  33. 

Arginin,    C6H14N402. — Arginin   is   the   most   widely   dis- 
tributed   of    the    decomposition    products    of    the    proteids. 


I  )!•:(■(  i. M  rnsi  I  lo\     I'KODUCTS    OF    PROTEIDS. 


79 


Every  proteid  so  far  subjected  to  decomposition  lias  yielded  this 
body  among  the  products.  Because  of  this  fact,  some  investi- 
gators consider  arginin  to  be  the  nucleus  of  the  proteid  mole- 
cule. Chemically,  arginin  is  guanidin-a-amino-valerianic  acid 
and  possesses  the  following  structural  formula: 

II    II    H    Nil 

I       I       I       I 
NH-C-C-C-C-COOH. 

I  I       I      I      I 

NH  =  C        II    H    H    H 

I 
NH2 

It  is  claimed  that  in  the  ordinary  metabolic  activities  of  the 
animal  body  arginin  gives  rise  to  urea. 

Histidin,  C6H9N302. — This  body  occurs  most  abundantly 
as  a  decomposition  product  of  globin,  the  proteid  constituent 

Fig.  34. 


Histiimx   Hydrochloride. 


of  haemoglobin.     The  free  base  is  laevorotatory,  whereas  the 
salts  of  histidin  are  dextro-rotatory.     Histidin  is  now  believed 


80  PHYSIOLOGICAL    CHEMISTRY. 

to  be  a-anrino-fi-imido-asol-propionic  acid  with  the  following 

structural  formula : 

H     NHo 

I       I 
HC  =  C-C-C-COOH. 

I      I 
H  H 

N\/NH 

CH 

Crystals  of  histidin  ltydrochloride  are  shown  in  Fig.  34,  p.  79. 

Experiments. 
While  the  ordinary  courses  in  physiological  chemistry  pre- 
clude any  extended  study  of  the  decomposition  products  of 
proteids,  the  manipulation  of  a  simple  decomposition  and  the 
subsequent  isolation  and  study  of  a  few  of  the  products  most 
easily  and  quickly  obtained  will  not  be  without  interest.  To 
this  end  the  student  may  use  the  following  decomposition  pro- 
cedure :  Treat  the  proteid  in  a  large  flask  with  water  contain- 
ing 3-5  per  cent  of  H2S04  and  place  it  on  a  water-bath  until 
the  proteid  material  has  been  decomposed  and  there  remains  a 
fine,  fluffy,  insoluble  residue.  Filter  off  this  residue  and 
neutralize  the  filtrate  with  Ba(OH)2  and  BaC03.  Filter  off 
the  precipitate  of  BaS04  which  forms  and  when  certain  that 
the  fluid  is  neutral  or  faintly  acid,1  concentrate  (first  on  a  wire 
gauze  and  later  on  a  water-bath)  to  a  syrup.  This  syrup 
contains  the  end-products  of  the  decomposition  of  the  proteid, 
among  which  are  proteoses,  peptones,  tyrosin,  leucin,  etc. 
Add  95  per  cent  alcohol  slowly  to  the  warm  syrup  until  no 
more  precipitate  forms,  stirring  continuously  with  a  glass  rod. 
This  precipitate  consists  of  proteoses  and  peptones.  Gather 
the  sticky  precipitate  on  the  rod  or  the  sides  of  the  dish  and, 
after  warming  the  solution  gently  for  a  few  moments,  filter 
it  through  a  filter  paper  which  has  not  been  previously  moist- 
ened.    After  dissolving  the  precipitate  of  proteoses  and  pep- 

1  If  the  solution  is  alkaline  in  reaction  at  this  point,  the  amino  acids  will 
be  broken  down  and  ammonia  will  be  evolved. 


DECOMPOSITION    PRODUCTS    OF    PROTEIDS.  8l 

tones  in  water1  the  solution  may  be  treated  according  to  the 
method  separation  given  on  page  59. 

Theleucin  and  tyrosin,  etc.,  are  in  solution  in  the  warm  alco- 
holic filtrate.  Concentrate  this  filtrate  on  the  water-hath  to  a 
thin  syrup,  transfer  it  to  a  Weaker,  and  allow  it  to  stand  over 
night  in  a  cool  place  for  crystallization.  The  tyrosin  first 
crystallizes  (Fig.  23,  page  68),  followed  later  hy  the  formation 
of  characteristic  crystals  of  impure  leucin  (see  Chapter  XIX). 
After  examining  these  crystals  under  the  microscope,  strain  off 
the  crystalline  material  through  fine  muslin,  heat  it  gently  in  a 
little  water  to  dissolve  the  leucin  (the  tyrosin  will  be  practically 
insoluble)  and  filter.  Concentrate  the  filtrate  and  allow  it  to 
stand  in  a  cool  place  over  night  for  the  crude  leucin  to  crystal- 
lize. Filter  off  the  crystals  and  use  them  in  the  tests  for  leucin 
given  on  page  82.  The  crystals  of  tyrosin  remaining  on  the 
paper  from  the  first  filtration  may  be  used  in  the  tests  for 
tyrosin  as  given  below.  If  desired,  the  tyrosin  and  leucin 
may  be  purified  by  recrystallizing  in  the  usual  manner.  Haber- 
mann  has  suggested  a  method  of  separating  leucin  and  tyrosin 
be  means  of  glacial  acetic  acid. 

Experiments  on  Tyrosin. 

Make  the  following  tests  with  the  tyrosin  crystals  already 
prepared  or  upon  some  pure  tyrosin  furnished  by  the  instruc- 
tor. 

1 .  Microscopical  Examination. — Place  a  minute  crystal  of 
tyri  isin  on  a  slide,  add  a  drop  of  water,  cover  with  a  cover  glass, 
and  examine  microscopically.  Now  run  more  water  under 
the  cover  glass  and  warm  in  a  bunsen  flame  until  the  tyrosin 
has  dissolved.  Allow  the  solution  to  cool  sloz^'ly  then  examine 
again  microscopically  and  compare  the  crystals  with  those 
shown  in  Fig.  23,  page  68. 

*At  this  point  the  aqueous  solution  of  the  proteoses  and  peptones  may 
be  filtered  to  remove  any  BaSCh  which  may  still  remain.  Tyrosin  crystals 
will  also  be  found  here,  since  it  is  less  soluble  than  the  leucin  and  may 
adhere  to  the  proteose-peptone  precipitate.  Add  the  crystals  of  tyrosin  to 
the  warm  alcohol  filtrate. 


82  PHYSIOLOGICAL    CHEMISTRY. 

2.  Solubility. — Try  the  solubility  of  very  small  amounts  of 
tyrosin  in  cold  and  hot  water,  cold  and  hot  95  per  cent  alcohol, 
dilute  NH4OH,  dilute  KOH  and  dilute  HC1. 

3.  Sublimation.- — Place  a  little  tyrosin  in  a  dry  test-tube, 
heat  gently  and  notice  that  the  material  does  not  sublime. 
How  does  this  compare  with  the  result  of  Experiment  3  under 
Leucin  ? 

4.  Hoffman's  Reaction. — This  is  the  name  given  to  Mil- 
Ion's  reaction  when  employed  to  detect  tyrosin.  Add  about 
3  c.c.  of  water  and  a  few  drops  of  Millon's  reagent  to  a  little 
tyrosin  in  a  test-tube.  Upon  dissolving  the  tyrosin  by  heat 
the  solution  gradually  darkens  and  may  assume  a  dark  red 
color.    What  group  does  this  test  show  to  be  present  in  tyrosin  ? 

5.  Piria's  Test. — Warm  a  little  tyrosin  on  a  watch  glass  on 
a  boiling  water-bath  for  20  minutes  with  3-5  drops  of  cone. 
PI0SO4.  Tyrosin-sulphuric  acid  is  formed  in  the  process. 
Cool  the  solution  and  wash  it  into  a  small  beaker  with  water. 
Now  add  CaC03  in  substance  slowly  with  stirring,  until 
the  reaction  of  the  solution  is  no  longer  acid.  Filter,  con- 
centrate the  filtrate  and  add  to  it  a  few  drops  (avoid  an  excess) 
of  very  dilute  neutral  ferric  chloride.  A  purple  or  violet  color, 
due  to  the  formation  of  the  ferric  salt  of  tyrosin-sulphuric  acid, 
is  produced.  This  is  one  of  the  most  satisfactory  tests  for 
the  identification  of  tyrosin. 

6.  Morner's  Test. — Add  about  3  c.c.  of  Morner's  reagent1 
to  a  little  tyrosin  in  a  test-tube,  and  gently  raise  the  tempera- 
ture to  the  boiling-point.    A  green  color  results. 

Experiments  on  Leucin. 

Make  the  following  tests  upon  the  leucin  crystals  already 
prepared  or  upon  some  pure  leucin  furnished  by  the  instructor. 

I,  2  and  3.  Repeat  these  experiments  according  to  the  direc- 
tions given  under  Tyrosin  (page  81). 

1  Morner's  reagent  is  prepared  by  thoroughly  mixing  1  volume  of  forma- 
lin, 45  volumes  of  distilled  water  and  55  volumes  of  concentrated  sulphuric 
acid. 


CHAPTER  V. 

GASTRIC    DIGESTION. 

Gastric  digestion  takes  place  in  the  stomach  and  is  pro- 
moted by  the  gastric  juice,  which  is  secreted  by  the  glands  of 
the  stomach  mucosa.  These  glands  are  of  two  kinds,  fundus 
glands  and  pyloric  glands  which  are  situated,  as  their  names 
imply,  in  the  regions  of  the  fundus  and  pylorus.  The  prin- 
cipal foods  acted  upon  in  gastric  digestion  are  the  proteids 
which  are  so  changed  by  its  processes  as  to  become  better 
prepared  for  further  digestion  in  the  intestine  and  for  their 
final  absorption. 

From  reliable  experiments  made  upon  lower  animals  it  is 
evident  that  the  gastric  juice  is  secreted  as  the  result  of  stimuli 
of  two  forms,  i.e.,  psychical  stimuli  and  chemical  stimuli. 
The  psychical  form  of  stimuli  may.  be  produced  by  the  sight, 
thought  or  taste  of  food,  and  the  chemical  stimuli  may  be  pro- 
duced by  certain  substances  such  as  water,  the  extractives  of 
meat,  etc.,  when  coming  in  contact  with  the  stomach  mucosa. 
The  volume  of  gastric  juice  secreted  during  any  given  period 
of  digestion,  varies  with  the  quantity  and  kind  of  the  food. 
These  conclusions  were  deduced  principally  from  a  series  of  so- 
called  delusive  feeding  experiments.  A  dog  was  prepared  with 
two  oesophageal  openings  and  a  gastric  fistula.  When  thus 
prepared  and  fed  foods  of  various  kinds  such  as  meat  and 
bread,  the  material  instead  of  passing  to  the  stomach,  would 
invariably  find  its  way  out  of  the  animal's  body  at  the  upper 
oesophageal  opening.  Through  the  medium  of  the  gastric 
fistula  the  course  of  the  secretion  of  gastric  juice  could  be 
carefully  followed.  It  was  found  that  when  the  dog  ate  meat, 
for  example,  there  was  a  large  secretion  of  gastric  juice  not- 
withstanding no  portion  of  the  food  eaten  had  reached  the 
stomach.     Further  experiments  made  through  the  medium  of 


84  PHYSIOLOGICAL    CHEMISTRY. 

a  cul-de-sac  formed  from  the  stomach  wall  have  given  us 
many  valuable  conclusions,  among  others  those  regarding  the 
influence  of  the  chemical  stimuli.  The  method  followed  was 
to  feed  the  animal  certain  substances  and  note  the  secretion 
of  gastric  juice  in  the  miniature  stomach  while  the  real  process 
of  digestion  was  taking  place  in  the  stomach  proper. 

Normal  gastric  juice  is  a  thin,  light  colored  fluid  which  is 
acid  in  reaction  and  has  a  specific  gravity  varying  between 
1. 001  and  1. 010.     It  contains  onry  2-3  per  cent  of  solid  mat- 
ter which  is  made  up  principally  of  HC1,  sodium  chloride, 
potassium  chloride,  earthy  phosphates,  mucin  and  the  enzymes 
pepsin,  rennin  and  probably  lipase;  the  HC1  and  the  enzymes 
are  of  the  greatest  importance.     The  acidity  of  the  gastric 
juice  is  due  to  free  hydrochloric  acid  which  is  secreted  by 
the  parietal  glands  of  the  fundus  and,  in  man,  is  generally 
present    to    the    extent    of    0.2-0.3    Per    cent-      When    the 
amount    of    HC1    varies    to    any    considerable    degree    from 
these  values   a  condition   of   hypoacidity  or  hyperacidity   is 
established.      Hydrochloric  acid  has   the  power  of   combin- 
ing with  proteid  substances  taken  in  the  food  forming  so- 
called  combined  hydrochloric  acid.     This  combined  acid  is  a 
less  potent  germicide  than  free  HC1  and  has  less  power  to 
destroy  the  amylolytic  enzyme  ptyalin  of  the  saliva.    This  last 
fact  explains  to  a  degree  the  possibility  of  the  continuance  of 
salivary  digestion  in  the  stomach.     The  HC1  of  the  gastric 
juice  forms  a  medium  in  which  the  pepsin  can  most  satisfac- 
torily digest  the  proteid  food,  and  at  the  same  time  it  acts  as 
an  antiseptic  or  germicide  which  prevents  putrefactive  proc- 
esses in  the  stomach.     It  also  possesses  the  power  of  inverting 
cane  sugar.    When  the  HC1  of  the  gastric  juice  is  diminished 
in  quantity   (hypoacidity)   or  absent,  as  it  may  be  in  many 
cases  of  functional  or  organic  disease,  there  is  no  check  to  the 
growth  of  micro-organisms  in  the  stomach.     There  are  how- 
ever  certain   of    the   more   resistant   spores    which    even   the 
normal  acidity  of  the  gastric  juice  will  not  destroy.     A  con- 
dition of  hypoacidity  may  also  give  rise  to  fermentation  with 
the  formation  of  such  bodies  as  lactic  acid  and  butyric  acid. 


GASTRIC    DIGESTION.  S5 

The  most  important  of  the  enzymes  of  the  gastric  juice 
is  the  proteolytic  enzyme  pepsin.  The  pepsin  does  not  origi- 
nate as  such  in  the  gastric  cells  hut  is  formed  from  it^  pre- 
cursor the  zymogen  or  mother-substance  pepsinogen  which  is 
produced  by  the  gastric  cells.  Upon  coming  in  contact  with 
the  HC1  of  the  secretion  this  pepsinogen  is  immediately 
transformed  into  pepsin.  Pepsin  is  not  active  in  alkaline  or 
neutral  solutions  but  requires  at  least  a  faint  acidity  before  it 
can  exert  its  power  to  dissolve  and  digest  proteids.  The  per- 
centage of  HC1  facilitating  the  most  rapid  peptic  action  varies 
with  the  character  of  the  proteid  acted  upon,  e.  g.,  0.08  per 
cent  to  0.1  per  cent  for  the  digestion  of  fibrin  and  0.25  per 
cent  for  the  digestion  of  coagulated  egg-white.  While  HC1 
is  the  acid  usually  employed  to  promote  artificial  peptic  pro- 
teolysis, other  acids,  organic  and  inorganic,  will  serve  the  same 
purpose.  Acidity  of  the  liquid  is  necessary  to  promote  the 
activity  of  the  pepsin,  but  the  acidity  need  not  necessarily  be 
confined  to  hydrochloric  acid. 

In  common  with  many  other  enzymes  pepsin  acts  best  at 
about  38°-40°  C.  and  its  digestive  power  decreases  as  the 
temperature  is  lowered,  the  enzyme  being  only  slightly  active 
at  o°  C.  Its  power  is  only  temporarily  inhibited  by  the  appli- 
cation of  such  low  temperatures,  however,  and  the  enzyme 
regains  its  full  proteolytic  power  upon  raising  the  temperature 
to  40 °  C.  As  the  temperature  of  a  digestive  mixture  is  raised 
above  40°C.  the  pepsin  gradually  loses  its  activity  until  at 
about  8o°-ioo°  C.  its  proteolytic  power  is  permanently  de- 
stroyed. 

The  products  of  peptic  proteolysis  are  acid  albuminate,  pro- 
teoses (albumoses)  and  peptones.  Only  a  comparatively  small 
amount  of  the  proteid  ingested  is  transformed  into  peptones, 
the  proteose  stage  being,  for  the  most  part,  the  final  stage  in 
peptic  proteolysis.  The  relative  amounts  of  proteoses  and  pep- 
tones formed  depends  to  a  great  extent  upon  the  character  of 
the  proteid  undergoing  digestion,  c.  g.,  a  greater  proportion  of 
proteoses  results  from  the  digestion  of  fibrin  than  from  the 


86  PHYSIOLOGICAL    CHEMISTRY. 

digestion  of  coagulated  egg-white.  Peptic  proteolysis  differs 
from  tryptic  proteolysis  (see  page  107)  in  that  the  former 
yields  larger  amounts  of  proteoses,  smaller  amounts  of  pep- 
tones and  no  considerable  quantity  of  crystalline  bodies  as 
end-products  in  the  brief  period  during  which  proteids  are 
ordinarily  subjected  to  gastric  digestion.  Prolonged  hydroly- 
sis with  gastric  juice  may  however,  yield  considerable  quan- 
tities of  the  non-proteid  end-products. 

Rennin,  the  second  enzyme  of  the  gastric  juice,  is  what  is 
known  as  a  milk  curdling  or  proteid  coagulating  enzyme. 
Rennin  acts  upon  the  caseinogen  of  the  milk,  splitting  it  into 
a  proteose-like  body  and  soluble  casein.  This  soluble  body,  in 
the  presence  of  calcium  salts,  combines  with  calcium  forming 
calcium  casein  or  true  casein  which  is  insoluble  and  precipi- 
tates. There  is  some  uncertainty  regarding  the  reaction,  to 
litmus,  in  which  rennin  shows  the  greatest  activity.  It  is, 
however,  usually  said  to  be  most  active  in  the  presence  of  a 
slight  acid  reaction,  as  would  naturally  be  expected.  It  is 
especially  abundant  in  the  gastric  mucosa  of  the  calf,  and  is 
used  to  curdle  the  milk  used  in  cheese  making.  Rennin  is 
always  present  normally  in  the  gastric  juice  but  in  certain 
pathological  conditions  such  as  atrophy  of  the  mucosa,  chronic 
catarrh  of  the  stomach  or  in  carcinoma  it  may  be  absent. 

Lipase  is  a  fat-splitting  enzyme  (seepage  97). 

PREPARATION  OF  AN  ARTIFICIAL  GASTRIC  JUICE. 

Dissect  the  mucous  membrane  of  a  pig's  stomach  from  the 
muscular  portion,  and  discard  the  latter.  Divide  the  mucous 
membrane  into  two  parts  (4/5  and  1/5).  Cut  up  the  larger 
portion,  place  it  in  a  large-sized  beaker  with  0.4  per  cent  HC1 
and  keep  at  38°-40°  C.  for  at  least  24  hours.  Filter  off  the 
residue,  consisting  principally  of  nuclein  and  anti-albumid,  and 
use  the  filtrate  as  an  artificial  gastric  juice.  This  filtrate  con- 
tains pepsin,  rennin  and  the  products  of  the  digestion  of  the 
stomach  tissue,  i.  e.,  acid  albuminate,  proteoses  and  peptones. 


gastric  digestion.  87 

Preparation  of  a  Glycerin  Extract  of  Pig's  Stomach. 
Take  the  one-fifth  portion  of  the  mucous  membrane  of  the 
pig's  stomach  not  used  in  the  preparation  of  the  artificial 
gastric  juice,  cut  it  up  very  finely,  place  it  in  a  small-sized 
beaker  and  cover  the  membrane  with  glycerin.  Stir  frequently 
and  allow  to  stand  at  room  temperature  for  at  least  24  hours. 
The  glycerin  will  extract  the  pepsinogen.  Separate,  with  a 
pipette  or  by  other  means,  the  glycerin  from  the  pieces  of 
mucous  membrane  and  use  the  glycerin  extract  as  required  in 
the  later  experiments. 

Products  of  Gastric  Digestion. 

Into  the  artificial  gastric  juice,  prepared  as  above  described, 
place  the  proteid  material  (fibrin,  coagulated  egg-white,  or 
lean  beef)  provided  for  you  by  the  instructor,  add  0.4  per 
cent  HC1  as  suggested  by  the  instructor  and  keep  the  diges- 
tion mixture  at  400  C.  for  2  to  3  days.  Stir  frequently  and 
keep  free  hydrochloric  acid  present  in  the  solution  (for  tests 
for  free  HC1  see  below). 

The  original  proteid  has  been  digested  and  the  solution  now 
contains  the  products  of  peptic  proteolysis,  i.  e.}  acid  albumin- 
ate, proteoses  and  peptones.  The  insoluble  residue  may  in- 
clude nuclein  and  anti-albumid.  Filter  the  digestive  mixture 
and  after  testing  for  free  HC1  neutralize  the  filtrate  with  KOH 
solution.  If  any  of  the  acid  albuminate  is  still  untrans formed 
into  proteoses  it  will  precipitate  upon  neutralization.  If  any 
precipitate  forms  heat  the  mixture  to  boiling,  and  filter.  If  no 
precipitate  forms  proceed  without  filtering. 

We  now  have  a  solution  containing  a  mixture  of  proteoses 
and  peptones.  Separate  and  identify  these  bodies  according 
to  the  directions  given  on  pages  59  and  60. 

Tests  for  Free  and  Combined  HC1. 

These  tests  are  made  with  a  class  of  reagents  known  as 
indicators,  so-called  because  they  serve  to  indicate  the  nature 
of  the  reaction  of  a  solution.     These  indicators  are  weak  acids 


88  PHYSIOLOGICAL    CHEMISTRY. 

or  bases  and  are  but  slightly  dissociable.  The  dissociation, 
with  the  formation  of  the  colored  ion,  forms  the  basis  for  the 
color  reaction. 

Examine  each  of  the  following"  solutions  by  means  of  the 
tests  given  below  and  report  the  results  in  a  form  similar 
to  the  chart  given  on  page  90:  (1)  0.2  per  cent  free  HC1. 
(2)  0.05  per  cent  free  HC1.  (3)  0.0 1  per  cent  free  HC1. 
(4)  0.05  per  cent  combined  HC1.  (5)  1  per  cent  lactic  acid. 
(6)  Equal  volumes  of  0.2  per  cent  free  HC1  and  1  per  cent 
lactic  acid.      (7)  1  per  cent  potassium  hydroxide. 

1.  Di-methyl-amino-azobenzene  (or  Topfer's  Reagent),1 


N(CH3)2  —  CCH4  —  N  =  N  —  C6H 


Place  1-2  drops  of  the  reagent  in  the  solution  to  be  tested. 
Free  mineral  acid  (HC1)  is  indicated  by  the  production  of  a 
pinkish-red  color.  If  free  acid  is  absent  a  yellow  color  ordi- 
narily results. 

2.  Giinzberg's  Reagent.2 — Place  1-2  drops  of  the  re- 
agent in  a  small  porcelain  evaporating  dish  and  carefully 
evaporate  to  dryness  over  a  low  flame.  Insert  a  glass  stirring 
rod  into  the  mixture  to  be  tested  and  draw  the  moist  end  of 
the  rod  through  the  dried  reagent.  Warm  again  gently  and 
note  the  production  of  a  purplish-red  color  in  the  presence  of 
free  HC1. 

3.  Boas'  Reagent.3 — Perform  this  test  in  the  same  manner 
as  2  above.  Free  hydrochloric  acid  is  indicated  by  the  pro- 
duction of  a  rose-red  color  which  becomes  less  pronounced  on 
cooling. 

4.  Congo  Red,4 

1  To   prepare    Topfer's    reagent    dissolve   0.5    gram   of   di-methyl-amino- 
azobenzene  in  100  c.c.  of  95  per  cent  alcohol. 

2  Giinzberg's  reagent  is  prepared  by  dissolving  2  grams  of  phloroglucin 
and  1  gram  of  vanillin  in  100  c.c.  of  95  per  cent  alcohol. 

3  Boas'   reagent   is  prepared  by   dissolving  5   grams   of   resorcin   and  3 
grams  of  saccharose  in  100  c.c.  of  95  per  cent  alcohol. 

4  This  indicator  is  prepared  by  dissolving  0.5  gram  of  congo  red  in  90 
c.c.  of  water  and  adding  10  c.c.  of  95  per  cent  alcohol.. 


GASTRIC    DIGESTION.  89 

S03Na 

VV  x '  x — x  \/\/ 

S03Na  NH2 

Conduct  this  test  according  to  the  directions  given  under  i  or  2 
page  88.  A  blue  color  indicates  free  HC1,  a  violet  color 
indicates  an  organic  acid  and  a  brown  color  indicates  com- 
bined HC1.  Congo  red  paper  made  by  immersing  ordinary 
filter  paper  in  the  indicator  and  subsequently  drying,  may  be 
used  in  this  test. 

5.  Tropseolin  OO,' 

NH(CCH5)— C0H4  — N  =  N— C6H4  — S03Na. 

Place  2  drops  of  the  solution  to  be  tested  and  1  drop  of  the 
indicator  in  an  evaporating  dish  and  evaporate  to  dryness 
over  a  low  flame.  The  formation  of  a  reddish-violet  color 
indicates  free  hydrochloric  acid. 

This  test  may  also  be  conducted  in  the  same  manner  as  2 
page  88. 

6.  Phenolphthalein,2 

/CeH.OH 

C— C(!H40H 

C 

xo 

Add  the  indicator  directly  to  the  solution,  or  apply  the  test 
according  to  the  directions  given  under  2  on  page  88.  This 
indicator  serves  to  denote  the  total  acidity  since  it  is  acted 
upon  by  free  mineral  acids,  combined  acids,  organic  acids  and 
acid  salts.     A  red  color  indicates  the  presence  of  an  alkali  and 

1  Prepared  by  dissolving  0.05  gram  of  tropseolin  00  in  100  c.c.  of  50  per 
cent  alcohol. 

2  This  indicator  is  prepared  by  dissolving  1  gram  of  phenolphthalein  in 
100  c.c.  of  95  per  cent  alcohol. 


9° 


PHYSIOLOGICAL    CHEMISTRY. 


the  indicator  is  colorless  in  the  presence  of  a  neutral  or  acid 
reaction.  This  indicator  is  unsatisfactory  in  the  presence  of 
ammonia. 

7.  Sodium  Alizarin  Sulphonate,1 

CO  (0H)2 

/       \        / 

C6H4  C6H 

\      /        \ 

CO  S03Na 

This  indicator  may  be  used  directly  in  the  solution  to  be  tested, 
or  the  test  may  be  applied  as  2,  page  88.  It  serves  to  indicate 
all  acid  reactions  except  those  due  to  combined  acids.  A 
reddish-violet  color  indicates  an  alkaline  reaction,  while  a  yel- 
low color  indicates  an  acid  reaction  due  to  a  free  mineral 
acid,  an  organic  acid  or  an  acid  salt. 

Report  the  results  of  your  tests  tabulated  in  the  form  given 
below : 


Name  of  Indicator. 

Solutions  Examined. 

0.2   <ft 

HCl. 

O.05  f 

HCl. 

O.OI  fc 

HCl. 

0.05$ 

Combined 

HCl. 

Lactic 
Acid. 

Equal  Vols. 
0.2  %  HCl 

and  1  % 
Lactic  Acid. 

KOH. 

Topfer'  s  Reagent. 

Giinzberg's  Reagent. 

Boas'  Reagent. 

Congo  Red. 

Tropseolin  00. 

Phenolphthalein. 

Alizarin. 

1  Prepare  this  indicator  by  dissolving  1   gram  of  sodium  alizarin  sul- 
phonate in  100  c.c.  of  water. 


GASTRIC    DIGESTION.  91 

GENERAL    EXPERIMENTS    ON    GASTRIC 
DIGESTION. 

1.  Conditions  Essential  for  the  Action  of  Pepsin. — Pre- 
pare four  test-tubes  as  follows : 

(a)  Five  c.c.  of  pepsin  solution. 

(b)  Five  c.c.  of  0.4  per  cent  HC1. 

(c)  Five  c.c.  of  pepsin-hydrochloric  acid  solution. 

(d)  Two  or  three  c.c.  of  pepsin  solution  and  2-3  c.c.  of  0.5 
per  cent  sodium  carbonate  solution. 

Introduce  into  each  tube  a  small  piece  of  fibrin  and  place 
them  on  the  water-bath  at  400  C.  for  one-halt  hour,  carefully 
noting  any  changes  which  occur.1  Now  combine  the  contents 
of  tubes  (a)  and  (b)  and  see  if  any  further  change  occurs 
after  standing  at  400  C.  for  15-20  minutes.  Explain  the 
results  obtained  from  these  five  experiments. 

2.  Influence  of  Different  Temperatures. — In  each  of  four 
test-tubes  place  5  c.c.  of  pepsin-hydrochloric  acid  solution. 
Immerse  one  tube  in  cold  water  from  the  faucet,  keep  a  second 
tube  at  room  temperature  and  place  a  third  on  the  water-bath 
at  40°  C.  Boil  the  contents  of  the  fourth  tube  for  a  few 
moments  then  cool  and  also  keep  it  at  400  C.  Into  each  tube 
introduce  a  small  piece  of  fibrin  and  note  the  progress  of 
digestion.  In  which  tube  does  the  most  rapid  digestion  occur? 
Explain  this. 

3.  The  Most  Favorable  Acidity. — Prepare  three  tubes  as 
follows : 

(a)  Five  c.c.  of  0.2  per  cent  pepsin-hydrochloric  acid  solu- 
tion. 

1  Digestion  of  fibrin  in  a  pepsin-hydrochloric  acid  solution  is  indicated 
first  by  a  swelling  of  the  proteid  due  to  the  action  of  the  acid,  and  later 
by  a  disintegration  and  dissolving  of  the  fibrin  due  to  the  action  of  the 
pepsin-hydrochloric  acid.  If  uncertain  at  any  time  whether  digestion  has 
taken  place,  the  solution  under  examination  may  be  filtered  and  the  biuret 
test  applied  to  the  filtrate.  A  positive  reaction  will  signify  the  presence 
of  acid  albuminate,  proteoses  (albumoses)  or  peptones,  the  presence  of  any 
one  of  which  would  indicate  that  digestion  has  taken  place. 


92  PHYSIOLOGICAL    CHEMISTRY. 

(b)  Two  or  three  c.c.  of  0.2  per  cent  HC1  -\-  1  c.c.  of  con- 
centrated HC1  -\-  5  c.c.  of  pepsin  solution. 

(c)  One  c.c.  of  0.2  per  cent  pepsin-hydrochloric  acid  solu- 
tion -f-  5  c.c.  of  water. 

Introduce  a  small  piece  of  fibrin  into  each  tube,  keep  them 
at  400  C,  and  note  the  progress  of  digestion.  In  which  de- 
gree of  acidity  does  the  fibrin  digest  the  most  rapidly? 

4.  Differentiation  Between  Pepsin  and  Pepsinogen. — 
Prepare  five  tubes  as  follows : 

(a)  Few  drops  of  glycerin  extract  of  pepsinogen  -|-  2-3 
c.c.  of  water. 

(b)  Few  drops  of  glycerin  extract  of  pepsinogen  -\-  5  c.c. 
of  0.2  per  cent  HC1. 

(c)  Few  drops  of  glycerin  extract  of  pepsinogen  -j-  5  c.c. 
of  0.5  per  cent  Na2COs. 

(d)  Two  or  three  c.c.  of  pepsin  solution  -(-  2-3  c.c.  of  1  per 
cent  Na2C03. 

(e)  Few  drops  of  glycerin  extract  of  pepsinogen  -\-  5  c.c. 
of  1  per  cent  Na2C03. 

Add  a  small  piece  of  fibrin  to  the  contents  of  each  tube, 
keep  the  five  tubes  at  400  C.  for  one-half  hour  and  observe 
any  changes  which  may  have  occurred.  To  (a)  add  an  equal 
volume  of  0.4  per  cent  HC1,  neutralize  (c),  (d)  and  (<?)  with 
HC1  and  add  an  equal  volume  of  0.4  per  cent  HC1.  Place 
these  tubes  at  40 °  C.  again  and  note  any  further  changes 
which  may  occur.  What  contrast  do  we  find  in  the  results 
from  the  three  last  tubes?    Why  is  this  so? 

5.  Comparative  Digestive  Power  of  Pepsin  with  Dif- 
ferent Acids. — Prepare  a  series  of  tubes  each  containing  one 
of  the  following  acids:  0.5  per  cent  acetic,  lactic,  oxalic  and 
butyric,  and  0.2  per  cent  hydrochloric,  sulphuric,  nitric  and 
combined  hydrochloric.  To  each  acid  add  a  few  drops  of  the 
glycerin  extract  of  pig's  stomach  and  a  small  piece  of  fibrin. 
Shake  well,  place  at  400  C.  and  note  the  progress  of  digestion. 
In  which  tubes  does  the  most  rapid  digestion  occur? 

6.  Influence  of  Metallic  Salts,  etc. — Prepare  a  series  of 


GASTRIC    DIGESTION.  93 

tubes  and  into  each  tube  introduce  4  c.c.  of  pepsin-hydrochloric 
acid  solution  and  }/2  c.c.  of  one  of  the  chemicals  listed  in 
Experiment  18  under  Salivary  Digestion,  page  40.     [ntroduce 

a  small  piece  of  fibrin  into  each  of  the  tubes  and  keep  them 
at  400  C.  for  one-half  hour.  Note  the  variations  in  the 
progress  of  digestion.  Where  has  the  least  rapid  digestion 
occurred  ? 

7.  Testing  the  Motor  and  Functional  Activities  of  the 
Stomach. — This  test  is  performed  the  same  as  Experiment  i<> 
under  Salivary  Digestion,  page  40.  If  the  experiment  was 
carried  out  under  salivary  digestion  it  will  not  be  necessary  to 
repeat  it  here. 

8.  Influence  of  Bile. — Prepare  five  tubes  as  follows : 

(a)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  -f-  J4— I 
c.c.  of  bile. 

(b)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  -j-  1—2 
c.c.  of  bile. 

(c)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  -f-  2-3 
c.c.  of  bile. 

(d)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  +  5  c.c. 
of  bile. 

(c)   Five  c.c.  of  pepsin-hydrochloric  acid  solution. 

Introduce  into  each  tube  a  small  piece  of  fibrin.  Keep  the 
tubes  at  400  C.  and  note  the  progress  of  digestion.  Does  the 
bile  exert  any  appreciable  influence?   How? 

9.  Influence  of  Rennin  on  Milk. — Prepare  a  series  of  five 
tubes  as  follows : 

(a)  Five  c.c.  of  fresh  milk  +  0.2  per  cent  HC1  (add  slowly 
until  precipitate  forms). 

(b)  Five  c.c.  of  fresh  milk  -f-  5  drops  of  rennin  solution. 

(c)  Five  c.c.  of  fresh  milk  -f-  10  drops  of  0.5  per  cent 
XaX03  solution. 

(d)  Five  c.c.  of  fresh  milk  -f-  10  drops  of  5  per  cent  am- 
monium oxalate  solution. 

(e)  Five  c.c.  of  fresh  milk  -(-  5  drops  of  0.2  per  cent  HC1. 
Xow  to  each  of  the  tubes  (c),  (d)  and  (e)  add  5  drops  of 


94  PHYSIOLOGICAL    CHEMISTRY. 

rennin  solution.  Place  the  whole  series  of  five  tubes  at  400  C. 
and  after  10-15  minutes  note  what  is  occurring  in  the  different 
tubes.     Give  a  reason  for  each  particular  result. 

10.  Tests  for  Lactic  Acid,  (a)  Uffelmann's  Reaction. — 
To  a  small  quantity  of  Uffelmann's  reagent1  in  a  test-tube  add 
a  few  drops  of  a  lactic  acid  solution.  The  amethyst-blue  color 
of  the  reagent  is  displaced  by  a  straw  yellow.  Other  organic 
acids  give  a  similar  reaction.  Mineral  acids  such  as  HC1  dis- 
charge the  blue  coloration  leaving  a  colorless  solution. 

(b)  Ferric  Chloride  Test. — Place  10  c.c.  of  very  dilute 
ferric  chloride  in  each  of  five  tubes.  To  the  first  add  2  c.c. 
of  0.2  per  cent  HC1,  to  the  second  2  c.c.  of  10  per  cent  alcohol, 
to  the  third  2  c.c.  of  2  per  cent  saccharose,  to  the  fourth  2  c.c. 
of  lactic  acid  and  to  the  fifth  2  c.c.  of  peptone  solution. 

It  is  evident  from  the  results  obtained  that  neither  of  the 
tests  given  above  is  satisfactory  for  the  detection  of  lactic  acid 
in  the  presence  of  other  substances  such  as  we  find  in  the 
gastric  contents. 

A  satisfactory  deduction  regarding  the  presence  of  lactic 
acid  can  only  be  made  after  extracting  the  gastric  contents 
with  ether,  evaporating  the  ether  extract  to  dryness  and  dis- 
solving the  residue  in  water.  This  residue  will  not  contain 
any  of  the  contaminations  which  interfered  with  the  simple 
tests  as  tried  above,  and  therefore  if  either  of  the  tests  is 
now  tried  on  the  dissolved  residue  of  the  ether  extract  we  may 
form  an  accurate  conclusion  regarding  the  presence  of  lactic 
acid. 

11.  Qualitative  Analysis  of  Stomach  Contents. — Take 
100  c.c.  of  stomach  contents  and  analyze  it  according  to  the 
following  scheme : 

1  Uffelmann's  reagent  is  prepared  by  adding  ferric  chloride  solution  to 
a  1  per  cent  solution  of  carbolic  acid  until  an  amethyst-blue  color  is 
obtained. 


GASTRIC    DIGESTION. 


95 


Stomach  Contents. 

Filter  and  test  the  filtrate  for  free  HC1. 

I 


Filtrate  I. 
Divide  into  two  parts. 
I 


Residue. 
Discard  after  making  a  micro- 
scopical examination. 


Filtrate  II. 
One-fifth  portion. 
Test  for: 

(a)  Pepsin. 

(b)  Bile  (see  p. 

(c)  Starch. 

(d)  Dextrin. 


Filtrate  III. 
Four-fifths  portion. 

Neutralize  carefully;   any  precipitate 
is  acid  albuminate.     If  a  precipitate 
-).  forms  filter  and  divide  the  filtrate 

into  tzvo  parts.  If  no  precipitate 
forms  divide  the  solution  into  two 
parts  without  filtering. 


Filtrate  IV. 
Two-thirds  portion. 
Heat    to    boiling    to     remove 
coagulable   proteids.     If   any 
precipitate  forms  filter  it  off; 
if  there  is  no  precipitate  pro- 
ceed directly  with  the  tests. 
Test  for: 
(a)   Sugar. 
{b)   Proteoses, 
(c)   Peptones. 


Filtrate  V. 
One-third  portion. 
Test  for : 

(a)  Lactic  acid. 
(&)  Rennin. 
(c)   Ptyalin. 


CHAPTER    VI. 
FATS. 

Fats  occur  very  widely  distributed  in  the  plant  and  animal 
kingdoms,  and  constitute  the  third  general  class  of  food  stuffs. 
In  plant  organisms  they  are  to  be  found  in  the  seeds,  roots  and 
fruit,  while  each  individual  tissue  and  organ  of  an  animal 
organism  contains  more  or  less  of  the  substance.  In  the 
animal  organism  fats  are  especially  abundant  in  the  bone 
marrow  and  adipose  tissue.  They  contain  the  same  elements 
as  the  carbohydrates,  i.  e.,  carbon,  hydrogen  and  oxygen, 
but  the  oxygen  is  present  in  smaller  percentage  than  in  the 
carbohydrates  and  the  hydrogen  and  oxygen  are  not  present 
in  the  proportion  to  form  water.     According  to  the  observa- 

Fig.  35- 


Beef  Fat.     {Long.) 

tions  of  Benedict  and  Osterberg  human  fat  contains  76.08  per 
cent  of  carbon  and  11.78  per  cent  of  hydrogen.  They  found 
the  heat  of  combustion  of  human  fat  to  be  9.523  calories  per 
gram'. 

96 


FATS.  97 

Chemically  considered  the  fats  are  esters1  of  the  tri-atomic 

alcohol,  glycerin,  and  the  mono-basic  fatty  acids.  The  II  of 
each  of  the  OH  groups  of  glycerin  is  replaced  by  a  fatty  acid 
radical  (see  page  65).     For  instance 

CH2-OH 

1 

CH  -OH 

I 
CH2-OH 

is  the  formula  for  glycerin  and  by  replacing  the  hydrogen 
of  the  hydroxyls  by  hydrocarbon  radicals  R,  R.'  and  R"  we 

obtain,  as  the  typical  formula  for  an  ordinary  neutral  fat, 

CH2-OOC-R 

I 
CH  -O-OC-R' 

I 
CH2- O-OC-R". 

The  positions  occupied  by  R,  R'  and  R"  in  the  above  formula 
may  be  filled  by  three  radicals  of  the  same  fatty  acid  or  by 
the  radicals  of  three  different  fatty  acids. 

By  hydrolysis  of  a  neutral  fat,  i.  c,  by  the  addition  to  the 
molecule  of  those  elements  which  are  eliminated  in  the  forma- 
tion of  the  fat  from  glycerin  and  fatty  acid,  it  may  be  resolved 
into  its  component  parts,  i.  c,  glycerin  and  fatty  acid.  In  the 
case  of  tri-palmitin  the  following  would  be  the  reaction : 

C3H5(0-C15H31CO)3  +  3H20  = 

TH-Palmiti,  C3H5(OH)3  +  3(C15H31COOH). 

Glycerin.  Palmitic  acid. 

This  process  is  called  saponification  and  may  be  produced  by 
boiling  with  alkalis;  by  the  action  of  steam  under  pressure; 
by  long-continued  contact  with  air  and  light;  by  the  action  of 
certain  bacteria  and  by  fat-splitting  enzymes  or  lipases,  c.  g., 

1  An  ester  is  an  ethereal  salt  consisting  of  an  organic  radical  united  with 
the  residue  of  an  inorganic  or  organic  acid. 


98  PHYSIOLOGICAL    CHEMISTRY. 

steapsin  (see  page  109).  The  cells  forming  the  walls  of  the 
intestines  evidently  possess  the  peculiar  property  of  synthesiz- 
ing the  glycerin  and  fatty  acid  thus  formed  so  that  after 
absorption  these  bodies  appear  in  the  blood  not  in  their  in- 
dividual forms  but  as  neutral  fats.  This  synthesis  is  similar 
to  that  enacted  in  the  absorption  of  proteid  material  where 
the  peptones  are  synthesized  into  albumin  in  the  act  of 
absorption. 

The  principal  animal  fats  with  which  we  have  to  deal  are 
stearin,  palmitin,  olein  and  butyrin.  Such  less  important 
forms  as  laurin  and  myristin  may  occur  abundantly  in  plant 
organisms.  The  generally  accepted  system  of  nomenclature 
for  these  fats  is  to  apply  the  prefix  "  tri  "  in  each  case  (e.  g., 
fn-palmitin)  since  three  fatty  acid  radicals  are  contained  in 
the  neutral  fat  molecule. 

Fats  occur  ordinarily  as  mixtures  of  several  individual  fats. 
For  example,  the  fat  found  in  animal  tissues  is  a  mixture  of 
tri-olein,  tri-palmitin  and  tri-stearin,  the  percentage  of  any  one 
of  these  fats  present  depending  upon  the  particular  species  of 
animal  from  whose  tissue  the  fat  was  derived.  Thus  the 
ordinary  mutton  fat  contains  more  tri-stearin  and  less  tri- 
olein than  the  pork  fat.  The  crystalline  forms  of  some  of 
the  more  common  fats  are  reproduced  in  Figs.  35,  36  and  37 
on  pages  96,  99  and  101.   . 

Pure  neutral  fats  are  odorless,  tasteless  and  generally  color- 
less. They  are  insoluble  in  the  ordinary  proteid  solvents  such 
as  water,  sodium  chloride  and  dilute  acids  and  alkalis  but  are 
very  readily  soluble  in  ether,  benzene,  chloroform  and  boiling 
alcohol.  Each  individual  fat  possesses  a  specific  melting-  or 
boiling-point  (according  to  whether  the  body  is  solid  or  fluid 
in  character)  and  this  property  of  melting  or  boiling  at  a 
definite  temperature  may  be  used  as  a  means  of  differentiation 
in  the  same  way  as  the  coagulation  temperature  (see  page  60) 
is  used  for  the  differentiation  of  coagulable  proteids.  When 
shaken  with  water,  or  a  solution  of  albumin,  soap  or  gum 


FATS.  99 

arable,  the  fats  are  finely  divided  and  assume  a  condition 
known  as  an  emulsion.  The  emulsion  with  water  is  transitory, 
while  the  emulsions  with  soap  or  albumin  solution  are  per- 
manent. 

The  fat  ingested  continues  essentially  unaltered  until  it 
reaches  the  intestines  where  it  is  acted  upon  by  steapsin  the 
fat-splitting  enzyme  of  the  pancreatic  juice  (see  page  109), 
and  glycerin  and  fatty  acid  are  formed  from  a  large  portion  of 
the  fat.  Part  of  the  fatty  acid  thus  formed  is  dissolved  in  the 
bile  and  absorbed  while  the  remainder  unites  with  the  alkalis 
of  the  pancreatic  juice  and  forms  soluble  soaps.  These  soaps 
may  further  act  to  produce  an  emulsion  of  the  remaining  fat 
and  thus  aid  in  its  absorption.  That  bile  is  of  assistance  in  the 
absorption  of  fat  is  indicated  by  the  increase  of  fat  in  the 
feces  when  for  any  reason  bile  does  not  pass  into  the  in- 
testines. 

The  fat  distributed  throughout  the  animal  body  is  formed 
partly  from  the  ingested  fat  and  partly  from  carbohydrates 

Fig.  36. 


Mutton  Fat.     (Long.) 


and  the  "  carbon  moiety  "  of  proteid  material.  The  formation 
of  adipoccre  and  the  occurrence  of  fatty  degeneration  are 
sometimes  given  as  proofs  of  the  formation  of  fat  from  pro- 


IOO  PHYSIOLOGICAL    CHEMISTRY. 

teid.  This  is  questioned  by  many  investigators.  Rather  more 
satisfactory  and  direct  proof  of  the  formation  of  fat  from 
proteid  material  has  been  obtained  by  experimentation  upon 
fix-maggots.  The  normal  content  of  fat  in  a  number  of 
maggots  was  determined  and  later  the  fat  content  of  others 
which  had  developed  in  blood  (84  per  cent  of  the  solid  matter 
of  blood  plasma  is  proteid  material)  was  determined.  The 
fat  content  was  found  to  have  increased  700  to  11 00  per  cent 
as  a  result  of  the  diet  of  blood  proteids.  Some  investigators 
are  not  inclined  to  accept  these  data  as  conclusive. 

Experiments  on  Fats. 

1.  Solubility. — Test  the  solubility  of  olive  oil  in  each  of 
the  ordinary  solvents  (see  page  4)  and  in  cold  alcohol,  hot 
alcohol,  chloroform  and  ether. 

2.  Formation  of  a  Transparent  Spot  on  Paper. — Place  a 
drop  of  olive  oil  upon  a  piece  of  ordinary  writing  paper.  Note 
the  transparent  appearance  of  the  paper  at  the  point  of  contact 
with  the  fat. 

3.  Reaction. — Try  the  reaction  of  fresh  olive  oil  to  litmus. 
Repeat  the  test  with  rancid  olive  oil.  What  is  the  reaction  of 
a  fresh  fat  and  how  does  this  reaction  change  upon  allowing 
the  fat  to  stand  for  some  time? 

4.  Formation  of  Acrolein. — To  a  little  olive  oil  in  a  mortar 
add  some  potassium  bisulphate,  KHS04,  and  rub  up  thor- 
oughly. Transfer  to  a  dry  test-tube  and  cautiously  heat. 
Note  the  irritating  odor  of  acrolein.  The  glycerin  of  the  fat 
has  been  dehydrolyzed  and  acrylic  aldehyde  or  acrolein  has 
been  produced.     This  is  the  reaction  which  takes  place : 

CH2-OH 

CH  •  OH  -»  CH2  =  CH  •  CHO  +  2H20. 

Acrolein. 

CH2-OH 

Glycerin. 


FATS. 


IOI 


5.  Emulsification. —  (a)  Shake  up  a  drop  of  neutral1  olive 
oil  with  a  little  water  in  a  test-tube.  The  fat  becomes  finely 
divided,  forming  an  emulsion.  This  is  not  a  permanent  emul- 
sion since  the  fat  separates  and  rises  to  the  top  upon  standing. 

(b)  To  5  c.c.  of  water  in  a  test-tube  add  2  or  3  drops  of  0.5 
per  cent  NaXO.j.  Introduce  into  this  faintly  alkaline  solution 
a  drop  of  neutral  olive  oil  and  shake.  The  emulsion  while  not 
permanent  is  not  so  transitory  as  in  the  ease  of  water  free 
from  sodium  carbonate. 

(c)  Repeat  (b)  using  rancid  olive  oil.  What  sort  of  an 
emulsion  do  you  get  and  why? 

( </ )  Shake  a  drop  of  neutral  olive  oil  with  a  dilute  albumin 
solution.  What  is  the  nature  of  this  emulsion?  Examine  it 
under  the  microscope. 

6.  Fat  Crystals. — Dissolve  a  small  piece  of  lard  in  ether  in 
a  test-tube,  add  an  equal  volume  of  alcohol  and  allow  the 
alcohol-ether  mixture  to  evaporate  spontaneously.     Examine 

Fig.  37- 


Pork  Fat. 


'Neutral  oli\e  oil  may  be  prepared  by  shaking  ordinary  olive  oil  with  a 
10  per  cent  solution  of  sodium  carbonate.  This  mixture  should  then  be 
extracted  with  ether  and  the  ether  removed  by  evaporation.  The  residue 
is  neutral  olive  oil. 


IQ2 


PHYSIOLOGICAL    CHEMISTRY. 


the  crystals  under  the  microscope  and  compare  them  with 
those  reproduced  in  Figs.  35,  36  and  37  on  pp.  96,  99  and  101. 
7.  Saponification  of  Bayberry  Tallow. — Fill  a  large  cas- 
serole two-thirds  full  of  water  rendered  strongly  alkaline  with 
solid  KOH  (a  stick  one  inch  in  length).  Add  about  10  grams 
of  bayberry  tallow  and  boil,  keeping  the  volume  constant  by 
adding  water  as  needed.  AVhen  saponification  is  complete1 
add  concentrated  HC1  slowly  until  no  further  precipitate  is 
produced.  Cool  the  solution  and  the  precipitate  of  free  fatty 
acid  will  rise  to  the  surface  and  form  a  cake.  In  this  in- 
stance the  fatty  acid  is  principally  palmitic  acid.  Remove  the 
cake,  break  it  into  small  pieces,  wash  it  with  water  by  decanta- 

Fig.  38. 


Palmitic  Acid. 


tion  and  transfer  to  a  small  beaker  by  means  of  95  per  cent 
alcohol.  Heat  on  a  water-bath  until  the  palmitic  acid  is  dis- 
solved, then  filter  through  a  dry  filter  paper  and  allow  the 
filtrate  to  cool  slowly  in  order  to  obtain  satisfactory  crystals. 
Write  the  reactions  which  have  taken  place  in  this  experiment. 
When  the  palmitic  acid  has  completely  crystallized  filter 

1  Place  2  or  3  drops  in  a  test-tube  full  of  water.     If  saponification  is 
complete  the  products  will  remain  in  solution. 


FATS. 


I03 


Fig.  39. 


o& 


off  the  alcohol,  dry  the  crystals  between  filter  papers  and  try 
the  te>t>  given  below. 

8.  Palmitic  Acid. —  (a)  Examine  the  crystals  under  the 
microscope  and  compare  them  with  those  shown  in  Fig.  38, 
opposite. 

(b)  Solubility. — Try  the  solubility  of  palmitic  acid  in  the 
same  solvents  as  used  on  fats  (see  p.  100). 

(cr)  Melting-Point. — Determine 
the  melting-point  of  palmitic  acid 
by  <»ne  of  the  methods  given  on 
pages  104  and  105. 

(d)  Formation  of  Transparent 
Spot  on  Paper, — Melt  a  little  of 
the  fatty  acid  and  allow  a  drop  to 
fall  upon  a  piece  of  ordinary  writ- 
ing paper.  How  does  this  com- 
pare with  the  action  of  a  fat  under 
similar  circumstances  ? 

(c)  Acrolein  Test. — Apply  the 
test  as  given  under  4,  page  100. 
Explain  the  result. 

9.  Saponification  of  Lard. — To  (wgj 
25  grams  of  lard  in  a  flask  add 
75  c.c.  of  alcoholic-potash  solution 
and  warm  upon  a  water-bath  until 
saponification  is  complete.  (This 
point  is  indicated  by  the  complete 
solubility  of  a  drop  of  the  solution 
when  allowed  to  fall  into  a  little 
water.)  Now  transfer  the  solu- 
tion from  the  flask  to  an  evapo- 
rating dish  containing  about  ioo 
c.c.  of  water  and  heat  on  a  water- 
bath  until  all  the  alcohol  has  been 

driven  off.     Precipitate  the  fatty  acid  with  HC1  and  cool  the 
solution.     Remove  the  fatty  acid  which  rises  to  the  surface, 


M  1:1. ting- Point  Apparatus. 


104  PHYSIOLOGICAL    CHEMISTRY. 

neutralize  the  solution  with  Na2COs  and  evaporate  to  dryness. 
Extract  the  residue  with  alcohol,  remove  the  alcohol  by  evapo- 
ration upon  a  water-bath  and  on  the  residue  of  glycerin  thus 
obtained  make  the  tests  as  given  below. 

10.  Glycerin,    (a)    Taste. — What  is  the  taste  of  glycerin? 

(b)  Solubility. — Try  the  solubility  of  glycerin  in  water, 
alcohol  and  ether. 

(c)  Acrolein  Test. — Repeat  the  test  as  given  under  4,  p.  100. 

(d)  Borax  Fusion  Test. — Fuse  a  little  glycerin  on  a  plat- 
inum wire  with  some  powdered  borax  and  note  the  charac- 
teristic green  flame.  This  color  is  due  to  the  glycerin  ester 
of  boric  acid. 

(e)  Fehlings  Test. — How  does  this  result  compare  with  the 
results  on  the  sugars  ? 

(/)  Solution  of  Cu(OH)2.— Form  a  little  Cu(OH)2  by 
mixing  CuS04  and  KOH.  Add  a  little  glycerin  to  this  sus- 
pended precipitate  and  note  what  occurs. 

11.  Melting-Point  of  Fat.  First  Method. — Insert  one  of 
the  melting-point  tubes,  furnished  by  the  instructor,  into  the 
liquid  fat  and  draw  up  the  fat  until  the  bulb  of  the  tube  is 
about  one-half  full  of  the  material.  Then  fuse  one  end  of  the 
tube  in  the  flame  of  a  bunsen  burner  and  fasten  the  tube  to  a 
thermometer  by  means  of  a  rubber  band  in  such  a  manner  that 
the  bottom  of  the  fat  column  is  on  a  level  with  the  bulb  of  the 
thermometer  (Fig.  39,  p.  103).  Fill  a  beaker  of  medium  size 
about  two-thirds  full  of  water  and  place  it  within  a  second 
larger  beaker  which  also  contains  water,  the  two  vessels  being 
separated  by  pieces  of  cork.  Immerse  the  bulb  of  the  ther- 
mometer and  the  attached  tube  in  such  a  way  that  the  bulb 
is  about  midway  between  the  upper  and  the  lower  surfaces  of 
the  water  of  the  inner  beaker.  The  upper  end  of  the  tube 
being  open  it  must  extend  above  the  surface  of  the  surround- 
ing water.  Apply  gentle  heat,  stir  the  water,  and  note  the 
temperature  at  which  the  fat  first  begins  to  melt.  This  point 
is  indicated  by  the  initial  transparency.  For  ordinary  fats, 
raise  the  temperature  very  cautiously  from  300  C.     To  deter- 


FATS.  105 

mine  the  congealing-poittt  remove  the  flame  and  note  the  tem- 
perature at  which  the  fat  begins  to  solidify.  Record  the  melt- 
ing-- and  congealing-points  of  the  various  fats  submitted  by  the 
instructor. 

Second  Method. — Fill  a  small  evaporating  dish  about  one- 
half  full  of  mercury  and  place  it  on  a  water-bath.  Put  a 
small  drop  of  the  fat  under  examination  on  an  ordinary 
cover  glass  and  place  this  upon  the  surface  of  the  mercury. 
Raise  the  temperature  of  the  water-bath  slowly  and  by  means 
of  a  thermometer  whose  bulb  is  immersed  in  the  mercury  note 
the  melting-point  of  the  fat.  Determine  the  congealing-point 
by  removing  the  flame  and  leaving  the  fat  drop  and  cover 
glass  in  position  upon  the  mercury.  How  do  the  melting- 
points  as  determined  by  this  method  compare  with  those  as 
determined  by  the  first  method?  Which  method  is  the  more 
accurate,  and  why? 


CHAPTER    VII. 
PANCREATIC    DIGESTION. 

As  soon  as  the  food  mixture  leaves  the  stomach  it  comes 
into  intimate  contact  with  the  bile  and  the  pancreatic  juice. 
Since  these  fluids  are  alkaline  in  reaction  there  can  obviously 
be  no  further  peptic  activity  after  they  have  become  intimately 
mixed  with  the  chyme  and  have  neutralized  the  acidity  pre- 
viously imparted  to  it  by  the  hydrochloric  acid  of  the  gastric 
juice.  The  pancreatic  juice  reaches  the  intestine  through  the 
duct  of  Wirsung  which  opens  into  the  intestine  near  the 
pylorus. 

Normally  the  secretion  of  pancreatic  juice  is  brought  about 
by  the  stimulation  produced  by  the  acid  chyme  as  it  enters  the 
duodenum.  This  secretion  is  probably  not  due  to  a  nervous 
reflex,  but  is  dependent  upon  the  presence,  in  the  epithelial  cells 
of  the  duodenum,  of  a  body  known  as  prosecretin.  This 
body  is  changed  into  secretin  through  the  hydrolytic  action 
of  the  acid  present  in  the  chyme.  The  secretin  is  then  ab- 
sorbed by  the  blood,  passes  to  the  pancreas  and  stimulates  the 
pancreatic  cells,  causing  a  flow  of  pancreatic  juice.  The  quan- 
tity of  juice  secreted  under  these  conditions  is  proportional  to 
the  amount  of  secretin  present.  The  activity  of  secretin  solu- 
tions is  not  diminished  by  boiling,  hence  the  body  does  not 
react  like  an  enzyme.  Further  study  of  the  body  may  show 
it  to  be  a  definite  chemical  individual  of  relatively  low  molec- 
ular weight.  It  has  not  been  possible  thus  far  to  obtain  secre- 
tin from  any  tissues  except  the  mucus  membrane  of  the  duo- 
denum and  jejunum. 

The  juice  as  obtained  from  a  permanent  fistula  differs 
greatly  in  its  properties  from  the  juice  as  obtained  from  a 
temporary  fistula,  and  neither  form  of  fluid  possesses  the  prop- 
erties of  the  normal  fluid.     Pancreatic  juice  collected   from 

1 06 


PANCREA1  [C    DIGESTION.  107 

a  natural  fistula  has  been  found  to  be  a  colorless,  clear,  strongly 
alkaline  fluid  which  foams  readily.  It  is  further  characterized 
by  containing  albumin  and  globulin  and  by  the  absence  of 

proteoses  and  peptone.  The  average  daily  secretion  ol  pan- 
creatic juice  is  C50  c.c.  and  its  specific  gravity  is  1.008.  The 
fluid  contain^  [.3  per  cent  of  solid  matter  and  the  freezing- 
point  is  —  0.47 °  C.  The  normal  pancreatic  secretion  contains 
at  least  four  distinct  enzymes.  They  are  trypsin,  a  proteolytic 
enzyme;  amylopsin,  an  amylolytic  enzyme;  steapsin,  a  fat- 
splitting  enzyme;  and  pancreatic  rennin,  a  milk-coagulating 
enzyme. 

The  most  important  of  the  four  enzymes  of  the  pancreatic 
juice  is  the  proteolytic  enzyme  trypsin.  This  enzyme  resem- 
bles pepsin  in  so  far  as  each  has  the  power  of  breaking  down 
proteid  material,  but  the  trypsin  has  much  greater  digestive 
power  and  is  able  to  cause  a  more  complete  decomposition  of 
the  complex  proteid  molecule.  In  the  process  of  normal  diges- 
tion the  proteid  constituents  of  the  diet  are  for  the  most  part 
transformed  into  proteoses  (albumoses)  before  coming  in  con- 
tact with  the  enzyme  trypsin.  This  is  not  absolutely  essential 
however,  since  trypsin  possesses  digestive  activity  sufficient  to 
transform  unaltered  native  proteids  and  to  produce  from  their 
complex  molecules  comparatively  simple  fragments.  Among 
the  products  of  tryptic  digestion  are  alkali  albuminate,  pro- 
teoses (albumoses),  peptone,  leucin,  tyrosin,  aspartic  acid, 
glutamic  acid,  lysiu,  histidin,  arginin,  tryptophan  and  am- 
monia. (The  crystalline  forms  of  many  of  these  products 
are  reproduced  in  Chapter  IV.)  Trypsin  does  not  occur  pre- 
formed in  the  gland,  but  exists  there  as  a  zymogen  called 
trypsinogen  which  bears  the  same  relation  to  trypsin  that 
pepsinogen  does  to  pepsin.  Trypsin  has  never  been  obtained 
in  a  pure  form  and  therefore  very  little  can  be  stated  definitely 
as  to  its  nature.  The  enzyme  is  the  most  active  in  alkaline 
solution  but  is  also  active  in  neutral  or  slightly  acid  solutions. 
Trypsin  is  destroyed  by  mineral  acids  and  may  also  be  de- 
stroyed by  comparatively  weak  alkali  {2  per  cent  sodium  car- 


108  PHYSIOLOGICAL    CHEMISTRY. 

bonate)  if  left  in  contact  for  a  sufficiently  long  time.  Tryp- 
sinogen, on  the  other  hand,  is  more  resistant  to  the  action  of 
alkalis. 

The  pancreatic  juice  which  is  collected  by  means  of  a  fistula 
possesses  practically  no  power  to  digest  proteid  matter.  A 
body  called  enterokinase  occurs  in  the  intestinal  juice  and  has 
the  power  of  converting  trypsinogen  into  trypsin.  This  proc- 
ess is  known  as  the  "  activation  "  of  trypsinogen  and  through 
it  a  juice  which  is  incapable  of  digesting  proteid  may  be  made 
active.  Enterokinase  is  not  always  present  in  the  intestinal 
juice  since  it  is  secreted  only  after  the  pancreatic  juice  reaches 
the  intestine.  It  resembles  the  enzymes  in  that  its  activity  is 
destroyed  by  heat,  but  differs  materially  from  this  class  of 
bodies  in  that  a  certain  quantity  of  intestinal  juice  is  capable 
of  activating  only  a  definite  quantity  of  trypsinogen.  Entero- 
kinase has  been  detected  in  the  higher  animals,  and  a  kinase 
possessing  similar  properties  has  been  shown  to  be  present  in 
bacteria,  fungi,  impure  fibrin,  lymph  glands  and  snake-venom. 
The  activation  of  trypsinogen  into  trypsin  may  be  brought 
about  in  the  gland  as  well  as  in  the  intestine  of  the  living 
organism  (Mendel  and  Rettger).  The  manner  of  the  activa- 
tion in  the  gland  and  the  nature  of  the  body  causing  it  are 
unknown  at  present. 

Amylopsin,  the  second  of  the  pancreatic  enzymes,  is  an 
amylolytic  enzyme  which  possesses  somewhat  greater  digestive 
power  than  the  ptyalin  of  the  saliva.  As  its  name  implies,  its 
activity  is  confined  to  the  starches,  and  the  products  of  its 
amylolytic  action  are  dextrins  and  sugars.  The  sugars  are 
principally  iso-maltose  and  maltose  and  these  by  the  further 
action  of  an  inverting  enzyme  are  partly  transformed  into 
dextrose. 

It  is  probable  that  the  saliva  as  a  digestive  fluid  is  not  abso- 
lutely essential.  The  ptyalin  is  destroyed  by  the  hydrochloric 
acid  of  the  gastric  juice  and  is  therefore  inactive  when  the 
chyme  reaches  the  intestine.  Should  undigested  starch  be 
present  at  this  point  however,  it  would  be  quickly  transformed 


PANCREATIC    DIGESTION.  109 

by  the  active  amylopsin.  This  enzyme  is  not  present  in  the 
pancreatic  juice  of  infants  during-  the  first  few  weeks  of  life, 
thus  showing  very  clearly  that  a  starchy  diet  is  not  normal 
for  this  period. 

It  has  been  claimed  that  amylopsin  has  a  slight  digestive 
action  upon  unboiled  starch. 

The  third  enzyme  of  the  pancreatic  juice  is  called  steapsin 
and  is  a  fat-Splitting  enzyme.  It  has  the  power  of  splitting 
the  neutral  fats  of  the  food  by  hydrolysis,  into  fatty  acid  and 
glycerin.     A  typical  reaction  would  be  as  follows : 

C3H5(0-C15H,1CO),  +  3H20  = 

Tri-Palmitin.  3(C15H31COOH)  +  C3H5(OH),. 

Palmitic  acid.  Glycerin. 

Recent  researches  make  it  probable  that  fats  undergo  saponi- 
fication to  a  very  large  extent  prior  to  their  absorption.  The 
fatty  acids  formed,  in  part  unite  with  the  alkalis  of  the  pan- 
creatic juice  and  intestinal  secretion  to  form  soluble  soaps ; 
in  part  they  are  doubtless  absorbed  dissolved  in  the  bile.  Some 
observers  believe  that  the  fats  may  also  be  absorbed  in  emul- 
sion— a  condition  promoted  by  the  presence  of  the  soluble 
soaps.  After  absorption  the  fatty  acids  are  re-synthesized  to 
form  neutral  fats  with  glycerin. 

Steapsin  is  very  unstable  and  is  easily  rendered  inert  by  the 
action  of  acid.  For  this  reason  it  is  not  possible  to  prepare 
an  extract  having  a  satisfactory  fat-splitting  power  from  a 
pancreas  which  has  been  removed  from  the  organism  for  a 
sufficiently  long  time  to  have  become  acid  in  reaction. 

The  fourth  enzyme  of  the  pancreatic  juice  is  called  pan- 
creatic rennin.  It  is  a  milk-coagulating  enzyme  whose  action 
is  very  similar  to  that  of  the  enzyme  rennin  found  in  the  gastric 
juice.  It  is  supposed  to  show  its  greatest  activity  at  a  tempera- 
ture varying  from  500  to  6o°  C. 


IIO  PHYSIOLOGICAL    CHEMISTRY. 

PREPARATION    OF    AN    ARTIFICIAL    PANCREATIC 

JUICE. 

After  removing  the  fat  from  the  pancreas  of  a  pig  or  sheep, 
finely  divide  the  organ  by  means  of  scissors  and  grind  it  in  a 
mortar.  If  convenient,  the  use  of  an  ordinary  meat  chopper 
is  a  very  satisfactory  means  of  preparing  the  pancreas. 

When  finely  divided  as  above  the  pancreas  should  be  placed 
in  a  500  c.c.  flask,  about  150  c.c.  of  30  per  cent  alcohol  added 
and  the  flask  and  contents  shaken  frequently  for  twenty-four 
hours.  (What  is  the  reaction  of  this  alcoholic  extract  at  the 
end  of  this  period,  and  why?)  Strain  the  alcoholic  extract 
through  cheese  cloth,  filter,  nearly  neutralize  with  KOH  solu- 
tion and  then  exactly  neutralize  it  with  0.5  per  cent  Na2COs. 

Products  of  Pancreatic  Digestion. 

Take  about  200  grams  of  lean  beef  which  has  been  freed 
from  fat  and  finely  ground  and  place  it  in  a  large-sized  beaker. 
Nearly  fill  the  beaker  with  pancreatic  extract  prepared  as 
above,  add  5  c.c.  of  an  alcoholic  solution  of  thymol  to  prevent 
putrefaction,  and  place  the  beaker  in  an  incubator  at  400  C. 
Stir  the  contents  of  the  beaker  frequently  and  add  more 
thymol  if  it  becomes  necessary.  Allow  digestion  to  proceed 
for  from  2  to  5  days  and  then  separate  the  products  formed 
as  follows :  Strain  off  the  undissolved  residue  through  cheese 
cloth,  nearly  neutralize  the  solution  with  dilute  hydrochloric 
acid  and  then  exactly  neutralize  it  with  0.2  per  cent  hydro- 
chloric acid.  A  precipitate  at  this  point  would  indicate  alkali 
albuminate.  Filter  off  any  precipitate  and  divide  the  filtrate 
into  two  parts,  a  one-fourth  and  a  three-fourth  portion. 

Transfer  the  one-fourth  portion  to  an  evaporating  dish  and 
make  the  separation  of  proteoses  and  peptones  as  well  as  the 
final  tests  upon  these  bodies  according  to  the  directions  given 
on  page  59. 

Place  about  5  c.c.  of  the  three-fourth  portion  in  a  test-tube 
and  add  about  1  c.c.  of  bromine  water.     A  violet  coloration 


PANCREATIC    DIGESTION.  HI 

indicates  the  presence  of  tryptophan  (see  page  77).  Concen- 
trate1 the  remainder  of  the  three-fourth  portion  to  a  thin  syrup 
and  make  the  separation  of  teuein  and  tyrosin  according  to  the 
directions  given  oil  page  81. 

GENERAL  EXPERIMENTS  ON  PANCREATIC 
DIGESTION. 

Experiments  on  Trypsin, 
t.  The  Most  Favorable  Reaction  for  Tryptic  Digestion. 
— Prepare  seven  tubes  as  follows : 

(a)  2-3  c.c.  of  neutral  pancreatic  extract  -f  2~3  cc-  of 
water. 

(b)  2-3  c.c.  of  neutral  pancreatic  extract  -f-  2-3  c.c.  of  1  per 
cent  Na2CO:i. 

(c)  2-3  c.c.  of  neutral  pancreatic  extract  +  2_3  cc-  °f  °-5 
per  cent  Na2C03. 

(d)  2-3  c.c.  of  neutral  pancreatic  extract  +  2-3  c.c.  of  0.2 
per  cent  HC1. 

(e)  2-3  c.c.  of  neutral  pancreatic  extract  +  2-3  c.c.  of  0.2 
per  cent  combined  HC1. 

('/)  2-3  c.c.  of  neutral  pancreatic  extract  +  2-3  c.c.  of  0.4 
per  cent  boric  acid. 

(g)  2-3  c.c.  of  neutral  pancreatic  extract  +  2-3  c.c.  of  0.4 
per  cent  acetic  acid. 

Add  a  small  piece  of  fibrin  to  the  contents  of  each  tube  and 
keep  them  at  400  C.  noting  the  progress  of  digestion.  In 
which  tube  do  we  find  the  most  satisfactory  digestion,  and 
why?  How  do  the  indications  of  the  digestion  of  fibrin  by 
trypsin  differ  from  the  indications  of  the  digestion  of  fibrin 
by  pepsin? 

2.  The  Most  Favorable  Temperature. —  (For  this  and 
the  following  series  of  experiments  under  tryptic  digestion 
use  the  neutral  extract  plus  an  equal  volume  of  0.5  per  cent 

1  If  the  solution  is  alkaline  in  reaction,  while  it  is  being  concentrated,  the 
amino  acids  will  be  broken  down  and  ammonia  will  be  liberated. 


112  PHYSIOLOGICAL    CHEMISTRY. 

Xa2C03.)  In  each  of  four  tubes  place  5  c.c.  of  alkaline  pan- 
creatic extract.  Immerse  one  tube  in  cold  water  from  the  fau- 
cet, keep  a  second  at  room  temperature  and  place  a  third  on  the 
water-bath  at  400  C.  Boil  the  contents  of  the  fourth  for  a  few 
moments,  then  cool  and  also  keep  it  at  400  C.  Into  each  tube 
introduce  a  small  piece  of  fibrin  and  note  the  progress  of 
digestion.  In  which  tube  does  the  most  rapid  digestion  occur? 
What  is  the  reason? 

3.  Influence  of  Metallic  Salts,  etc. — Prepare  a  series  of 
tubes  and  into  each  tube  place  6  volumes  of  water,  3  volumes  of 
alkaline  pancreatic  extract  and  1  volume  of  one  of  the  chemicals 
listed  in  Experiment  18  under  Salivary  Digestion,  page  40. 

Introduce  a  small  piece  of  fibrin  into  each  of  the  tubes  and 
keep  them  at  40 °  C.  for  one-half  hour.  Shake  the  tubes  fre- 
quently.    In  which  tubes  do  we  get  the  least  digestion? 

4.  Influence  of  Bile. — Prepare  five  tubes  as  follows : 

(a)  Five  c.c.  of  pancreatic  extract  +  j4-i  c.c.  of  bile. 

(b)  Five  c.c.  of  pancreatic  extract  -j-  1-2  c.c.  of  bile. 

(c)  Five  c.c.  of  pancreatic  extract  -j-  2-3  c.c.  of  bile. 

(d)  Five  c.c.  of  pancreatic  extract  +  5  c.c.  of  bile. 

(e)  Five  c.c.  of  pancreatic  extract. 

Introduce  into  each  tube  a  small  piece  of  fibrin  and  keep 
them  at  400  C.  Shake  the  tubes  frequently  and  note  the  prog- 
ress of  digestion.  Does  the  presence  of  bile  retard  tryptic 
digestion?  How  do  these  results  agree  with  those  obtained 
under  gastric  digestion? 

Experiments  on  Amylopsin. 
1.  The  Most  Favorable  Reaction. — Prepare  seven  tubes 
as  follows : 

(a)  One  c.c.  of  neutral  pancreatic  extract  -j-  1  c.c.  of  starch 
paste  +  2  c.c.  of  water. 

(b)  One  c.c.  of  neutral  pancreatic  extract  -j-  1  c.c.  of  starch 
paste  -f-  2  c.c.  of  1  per  cent  Na2COs. 

(c)  One  c.c.  of  neutral  pancreatic  extract  +  1  c-c-  °f  starch 
paste  -f-  2  c.c.  of  0.5  per  cent  Na2C03. 


PANCREATIC    DIGESTION.  1 13 

(d)  One  c.c.  of  neutral  pancreatic  extract  -\-  J  cc-  °f  starch 
paste  +  2  c.c.  of  0.2  per  cent  HC1. 

(e)  One  c.c.  of  neutral  pancreatic  extract  -f-  1  c.c.  of  starch 
paste  -f-  2  c.c.  of  0.2  per  cent  combined  HC1. 

(/)  One  c.c.  of  neutral  pancreatic  extract  -\-  1  c.c.  of  starch 
paste  +  2  c.c.  of  0.4  per  cent  boric  acid. 

(g)  One  c.c.  of  neutral  pancreatic  extract  -(-  1  c.c.  of  starch 
paste  -j-  2  c.c.  of  0.4  per  cent  acetic  acid. 

Shake  each  tube  thoroughly  and  place  them  on  the  water- 
bath  at  400  C.  At  the  end  of  a  half-hour  divide  the  contents 
of  each  tube  into  two  parts  and  test  one  part  by  the  iodine  test 
and  the  other  part  by  Fehling-'s  test.  Where  do  you  find  the 
most  satisfactory  digestion?  How  do  the  results  here  com- 
pare with  those  obtained  from  the  similar  series  under  Trypsin, 
page  in. 

2.  The  Most  Favorable  Temperature. —  (For  this  and 
the  following  series  of  experiments  upon  amylopsin  use  the 
neutral  extract  plus  an  equal  volume  of  0.5  per  cent  Xa2CO;!.) 
In  each  of  four  tubes  place  2-3  c.c.  of  alkaline  pancreatic  ex- 
tract. Immerse  one  tube  in  cold  water  from  the  faucet,  keep  a 
second  at  room  temperature  and  place  a  third  on  the  water- 
bath  at  400  C.  Boil  the  contents  of  the  fourth  for  a  few 
moments,  then  cool  and  also  keep  it  at  400  C.  Into  each  tube 
introduce  2-3  c.c.  of  starch  paste  and  note  the  progress  of 
digestion.  At  the  end  of  one-half  hour  divide  the  contents  of 
each  tube  into  two  parts  and  test  one  part  by  the  iodine  test 
and  the  other  part  by  Fehling's  test.  In  which  tube  do  you 
find  the  most  satisfactory  digestion?  How  does  this  result 
compare  with  the  result  obtained  in  the  similar  series  of  experi- 
ments under  Trypsin  (see  page  11 1)  ? 

3.  Influence  of  Metallic  Salts,  etc. — Prepare  a  series  of 
tubes  and  into  each  tube  place  3  volumes  of  water,  3  volumes  of 
alkaline  pancreatic  extract,  1  volume  of  one  of  the  chemicals 
listed  in  Experiment  18  under  Salivary  Digestion,  page  40. 
and  3  volumes  of  starch  paste.  Be  sure  to  introduce  the. starch 
paste  into  the  tube  last.     Why?     Shake  the  tubes  well  and 

9 


114  PHYSIOLOGICAL    CHEMISTRY. 

place  them  on  the  water-bath  at  400  C.  At  the  end  of  a  half- 
hour  divide  the  contents  of  each  tube  into  two  parts  and  test 
one  part  by  the  iodine  test  and  the  other  part  by  Fehling's  test. 
What  are  your  conclusions? 

4.  Influence  of  Bile. — Prepare  five  tubes  as  follows : 

(a)  2-3  c.c.  of  pancreatic  extract  +  2-3  c.c.  of  starch  paste 
+  y2-i  c.c.  of  bile. 

(b)  2-3  c.c.  of  pancreatic  extract  +  2_3  c-c  of  starch  paste 
-f-  1-2  c.c.  of  bile. 

(c)  2-3  c.c.  of  pancreatic  extract  -\-  2-3  c.c.  of  starch  paste 
-f-  2-3  c.c.  of  bile. 

(d)  2-3  c.c.  of  pancreatic  extract  -\-  2-3  c.c.  of  starch  paste 
+  5  c.c.  of  bile. 

(e)  2-3  c.c.  of  pancreatic  extract  -j-  2-3  c.c.  of  starch  paste. 

Shake  the  tubes  thoroughly  and  place  them  on  the  water- 
bath  at  40 °  C.  Note  the  progress  of  digestion  frequently  and 
at  the  end  of  a  half-hour  divide  the  contents  of  each  tube  into 
two  parts  and  test  one  part  by  the  iodine  test  and  the  other  part 
by  Fehling's  test.  What  are  your  conclusions  regarding  the 
influence  of  bile  upon  the  action  of  amylopsin? 

5.  Digestion  of  Dry  Starch. — To  a  little  dry  starch  in  a 
test-tube  add  about  5  c.c.  of  pancreatic  extract  and  place  the 
tube  on  the  water-bath  at  400  C.  At  the  end  of  a  half  hour 
filter  and  test  separate  portions  of  the  fil.trate  by  the  iodine  and 
Fehling  tests.  What  do  you  conclude  regarding  the  action  of 
amylopsin  upon  dry  starch?  Compare  this  result  with  that 
obtained  in  the  similar  experiment  under  Salivary  Digestion 
(page  38). 

6.  Digestion  of  Inulin. — To  5  c.c.  of  inulin  solution  in  a 
test-tube  add  10  drops  of  pancreatic  extract  and  place  the  tube 
on  the  water-bath  at  400  C.  After  one-half  hour  test  the 
solution  by  Fehling's  test.1  Is  any  reducing  substance  present? 
What  do  you  conclude  regarding  the  digestion  of  inulin  by 
amylopsin? 

1  If  the  inulin  solution  gives  a  reduction  before  being  acted  upon  by  the 
pancreatic  juice,  it  will  be  necessary  to  determine  the  extent  of  the  original 
reduction  by  means  of  a  "check"  test  (see  page  26). 


pancreatic  digestion.  1 15 

Experiments  on  Steapsix. 

i.  "Litmus-Milk"  Test. — Into  each  of  two  test-tubes  in- 
troduce 10  c.c.  of  milk  and  a  small  amount  of  litmus  powder. 
To  the  contents  of  one  tube  add  3  c.c.  of  neutral  pancreatic 
extract  and  to  the  contents  of  the  other  tube  add  3  c.c.  of  water 
or  of  boiled  neutral  pancreatic  extract.  Keep  the  tubes  at  400 
C.  and  note  any  changes  which  may  occur.  What  is  the 
result  and  how  do  you  explain  it? 

2.  Ethyl  Butyrate  Test. — Into  each  of  two  test-tubes  intro- 
duce 4  c.c.  of  water,  2  c.c.  of  ethyl  butyrate,  CaH7COO"  C2H5, 
and  a  small  amount  of  litmus  powder.  To  the  contents  of  one 
tube  add  4  c.c.  of  neutral  pancreatic  extract  and  to  the  contents 
of  the  other  tube  add  4  c.c.  of  water  or  bailed  neutral  pan- 
creatic extract.  Keep  the  tubes  at  40°  C.  and  observe  any 
changes  which  may  occur.  What  is  the  result  and  how  do 
you  explain  it?  Write  the  equation  for  the  reaction  which 
has  taken  place. 

Experiments  on  Pancreatic  Rennin. 
Prepare  two  test-tubes  as  follows : 

(a)  Five  c.c.  of  milk  +  10  drops  of  pancreatic  extract. 

(b)  Five  c.c.  of  milk  +  20  drops  of  pancreatic  extract. 
Place  the  tubes  at  40°-45°    C.    for  a  half  hour  without 

shaking.  Xote  the  formation  of  a  clot.1  How  does  the  action 
of  pancreatic  rennin  compare  with  the  action  of  the  gastric 
rennin  ? 

'This  reaction  will  not  always  succeed,  owing  to  conditions  which  are 
not  well  understood. 


CHAPTER    VIII. 
BILE. 

The  bile  is  secreted  continuously  by  the  liver  and  passes  into 
the  intestine  through  the  common  bile  duct  which  opens  near 
the  pylorus.  Bile  is  not  secreted  continuously  into  the  intes- 
tine. In  a  fasting  animal  no  bile  enters  the  intestine,  but 
when  food  is  taken  the  bile  begins  to  flow ;  the  length  of  time 
elapsing  between  the  ingestion  of  the  food  and  the  secretion  of 
the  bile  as  well  as  the  qualitative  and  quantitative  character- 
istics of  the  secretion  depending  upon  the  nature  of  the  food 
ingested.  Fats,  the  extractives  of  meat  and  the  end-products 
of  gastric  digestion  (acid  albuminate,  proteoses  and  peptones) 
cause  a  copious  secretion  of  bile,  whereas  such  substances  as 
water,  acids  and  boiled  starch  paste  fail  to  do  so.  In  general 
a  rich  proteid  diet  is  supposed  to  increase  the  amount  of  bile 
secreted,  whereas  a  carbohydrate  diet  would  rather  tend  to 
decrease  the  amount. 

We  may  look  upon  the  bile  as  an  excretion  as  well  as  a 
secretion.  In  the  fulfillment  of  its  excretory  function  it  passes 
such  bodies  as  lecithin,  metallic  substances  and  cholesterin  into 
the  intestine  and  in  this  way  aids  in  removing  them  from  the 
organism.  The  bile  assists  materially  in  the  absorption  of 
fats  from  the  intestine  by  its  solvent  action  on  the  fatty  acids 
formed  by  the  action  of  the  pancreatic  juice. 

The  bile  is  a  very  thick,  viscid  substance  which  is  alkaline 
in  reaction  to  litmus  and  ordinarily  possesses  a  decidedly 
bitter  taste.  It  varies  in  color  in  the  different  animals,  the 
principal  variations  being  yellow,  brown  and  green.  Fresh 
human  bile  ordinarily  has  a  green  or  golden-yellow  color. 
Post-mortem  bile  is  variable  in  color.  It  is  very  difficult  to 
determine  accurately  the  amount  of  normal  bile  secreted  dur- 
ing any  given  period.    For  an  adult  man  it  has  been  variously 

116 


BILE.  I  I  7 

estimated  at  from  500  c.c.  to  cioo  c.c.  for  twenty  four  hours. 
The  specific  gravity  of  the  bile  varies  bet  \\  een  [.010  and  1 .040, 
and  the  freezing-point  is  about  — 0.56  C.  As  secreted  by 
the  liver,  the  bile  is  a  clear,  limpid  fluid  which  contains  a  rela- 
tively low  contenl  of  solid  matter.  Such  bile  would  ha 
specific  gravity  of  approximately  r.oio.  After  it  reaches  the 
gall  Madder,  however,  it  becomes  mixed  with  mucous  material 
from  the  walls  of  the  gall-bladder,  and  this  pro*  npled 

with  the  continuous  absorption  of  water  from  the  bile  has  a 
tendency  to  concentrate  the  secretion.  Therefore  the  bile,  as 
we  find  it  in  the  gall-blaader,  ordinarily  possesses  a  higher 
specific  gravity  than  that  of  the  freshly  secreted  fluid.  The 
specific  gravity  under  these  conditions  may  run  as  high  as 
1.040. 

The  principal  constituents  of  the  bile  are  the  salts  of  the 
bile  acids,  bile  pigments,  neutral  fats,  lecitJiin  and  cholesterin, 
besides  the  salts  of  iron,  copper,  calcium  and  magnesium. 
Zinc  has  also  frequently  been  found  in  traces. 

The  bile  acids,  which  are  elaborated  exclusively  by  the 
hepatic  cells,  may  be  divided  into  two  groups,  the  glycocholic 
acid  group  and  the  taurocholic  acid  group.  In  human  bile 
glycocholic  acid  predominates,  while  taurocholic  acid  is  the 
more  abundant  in  the  bile  of  carnivora.  The  bile  acids  are 
conjugate  amino-acids,  the  glycocholic  acid  yielding  glycocoll, 

CPL  •  NH„ 

I 
COOH, 

and  cholic  acid  upon  decomposition,  whereas  taurocholic  acid 

gives  rise  to  tauriu, 

CHo  •  NH2 

I 

CH2  •  S02  •  OH, 

and  cholic  acid  under  like  conditions.  Glycocholic  acid  con- 
tains some  nitrogen  but  no  sulphur,  whereas  taurocholic  acid 
contains  both  these  elements.     The  sulphur  of  the  taurocholic 


n8 


PHYSIOLOGICAL    CHEMISTRY. 


acid  is  present  in  the  taurin  (amino-ethyl-sulphonic  acid),  of 
which  it  is  a  characteristic  constituent.  There  are  several 
varieties  of  cholic  acid  and  therefore  we  have  several  forms 
of  glycocholic  and  taurocholic  acids,  the  variation  in  consti- 
tution depending  upon  the  nature  of  the  cholic  acid  which 
enters  into  the  combination.  The  bile  acids  are  present  in  the 
bile  as  salts  of  one  of  the  alkalis,  generally  sodium.  The  sodi- 
um glycocholate  and  sodium  taurocholate  may  be  isolated  in 
crystalline  form,  either  as  balls  or  rosettes  of  fine  needles  or  in 
the  form  of  prisms  having  ordinarily  four  or  six  sides  (Fig. 
40,  below).  The  salts  of  the  bile  acids  are  dextro-rotatory. 
Among  other  properties  these  salts  have  the  power  of  holding 
the  cholesterin  and  lecithin  of  the  bile  in  solution. 

Fig.  40. 


Bile  Salts. 


The  bile  pigments  are  important  and  interesting  biliary  con- 
stituents. The  following  have  been  isolated :  bilirubin,  bili- 
verdin,  bilifuscin,  biliprasin,  bilihumin,  bilicyanin  and  chole- 
telin.  Of  these,  bilirubin  and  biliverdin  are  the  most  important 
and  predominate  in  normal  bile.  Bilirubin  may  be  isolated  as 
a  reddish-yellow  powder  and  biliverdin  may  be  obtained  in  the 
form  of  a  green  powder.    The  colors  possessed  by  the  various 


BILE.  119 

varieties  of  normal  bile  are  due  almosl  entirely  to  these  two 
pigments,  the  biliverdin  being  the  predominant  pigment  in 
greenish  bile  and  the  bilirubin  being  the  principal  pigment  in 
lighter  colored  bile.  The  pigments,  other  than  the  two  just 
mentioned,  have  been  found  almost  exclusively  in  biliary  cal- 
culi or  in  altered  bile  obtained  at  post-mortem  examinations. 

Bilirubin,  which  is  perhaps  the  most  important  of  the  bile 
pigments,  is  apparently  derived  from  the  blood  pigment,  the 
iron  freed  in  the  process  being  held  in  the  liver.  Bilirubin 
has  the  same  percentage  composition  as  haematoporphyrin, 
which  may  be  produced  from  haematin.  It  is  a  specific  product 
of  the  liver  cells  but  may  also  be  formed  in  other  parts  of  the 
body.  The  pigment  may  be  obtained,  in  part,  in  the  form 
of  reddish-yellow   rhombic  plates    (Fig.  41,  below)    upon  the 

Fig.  41. 


Bilirubin    (H^matoidin).      (Ogdcn.) 

spontaneous  evaporation  of  its  chloroform  solution.  The 
crystalline  form  of  bilirubin  is  practically  the  same  as  that 
of  hsematoidin.  Tt  is  easily  soluble  in  chloroform,  somewhat 
less  soluble  in  alcohol  and  only  slightly  soluble  in  ether  and 
benzene.  Bilirubin  has  the  power  of  combining  with  certain 
metals,  particularly  calcium,  to  form  combinations  which  are 
no  longer  soluble  in  the  solvents  of  the  unaltered  pigment. 
Upon  long  standing  in  contact  with  the  air,  the  reddish-yellow 
bilirubin  is  oxidized  with  the  formation  of  the  green  biliverdin. 
Bilirubin  occurs  in  animal  fluids  as  soluble  bilirubin-alkali. 


120  PHYSIOLOGICAL    CHEMISTRY. 

Solutions  of  bilirubin  exhibit  no  absorption-bands.  If  an 
ammoniacal  solution  of  bilirubin-alkali  in  water  is  treated 
with  a  solution  of  zinc  chloride,  however,  it  shows  bands 
similar  to  those  of  bilicyanin  (Absorption  Spectra,  Plate  II), 
the  two  bands  between  C  and  D  being  rather  well  defined. 

Biliverdin  is  particularly  abundant  in  the  bile  of  herbivora. 
It  is  soluble  in  alcohol  and  glacial  acetic  acid  and  insoluble  in 
water,  chloroform  and  ether.  Biliverdin  is  formed  from 
bilirubin  upon  oxidation.  It  is  an  amorphous  substance,  and 
in  this  differs  from  bilirubin  which  may  be  at  least  partly 
crystallized  under  proper  conditions.  In  common  with  bili- 
rubin, it  may  be  converted  into  hydrobilirubin  by  nascent 
hydrogen. 

The  neutral  solution  'of  bilicyanin  or  cholecyanin  is  bluish- 
green  or  steel-blue  and  possesses  a  blue  fluorescence,  the  alka- 
line solution  is  green  with  no  appreciable  fluorescence  and  the 
strongly  acid  solution  is  violet-blue.  The  alkaline  solution 
exhibits  three  absorption-bands,  the  first  a  dark,  well-defined 
band  between  C  and  D  somewhat  nearer  C;  the  second  a  less 
sharply-defined  band  extending  across  D  and  the  third  a  rather 
faint  band  between  E  and  F,  near  E  (Absorption  Spectra, 
Plate  II).  The  strongly  acid  solution  exhibits  two  absorption 
bands,  both  lying  between  C  and  E  and  separated  by  a  narrow 
space  near  D.  A  third  band,  exceedingly  faint,  may  ordinarily 
be  seen  between  b  and  F. 

Biliary  calculi,  otherwise  designated  as  biliary  concretions 
or  gall  stones,  are  frequently  formed  in  the  gall-bladder. 
These  deposits  may  be  divided  into  three  classes,  cholcsterin 
calculi,  pigment  calculi  and  calculi  made  up  almost  entirely  of 
inorganic  material.  This  last  class  of  calculus  is  formed  prin- 
cipally of  the  carbonate  and  phosphate  of  calcium  and  is 
rarely  found  in  man  although  quite  common  to  cattle.  The 
pigment  calculus  is  also  found  in  cattle,  but  is  more  common 
to  man  than  the  inorganic  calculus.  This  pigment  calculus 
ordinarily  consists  principally  of  bilirubin  in  combination  with 
calcium;  biliverdin  is  sometimes  found  in  small  amount.     The 


BILE.  I  2  I 

cholesterin  calculus  is  the  one  found  most  frequently  in  man. 
These  may  be  formed  almost  entirely  of  cholesterin,  in  which 
event  the  color  of  the  calculus  is  very  light,  or  they  may  con- 
tain more  or  less  pigment  and  inorganic  matter  mixed  with  the 
cholesterin,  which  tends  to  give  us  calculi  of  various  colors. 
For  discussion  of  cholesterin  see  page  222. 

Experiments  on  Bile. 

1.  Reaction. — Test  the  reaction  of  fresh  ox  bile  to  litmus. 

2.  Nucleo-proteid. — Acidify  a  small  amount  of  hile  with 
dilute  acetic  acid.     A  precipitate  of  nucleo-proteid  forms. 

3.  Inorganic  Constituents. — Test  for  chlorides,  sulphates 
and  phosphates  (see  page  37). 

4.  Tests  for  Bile  Pigments,  (a)  Gwielin's  Test. — To 
about  5  c.c.  of  concentrated  nitric  acid  in  a  test-tube  add  2-3 
c.c.  of  diluted  bile  carefully  so  that  the  two  fluids  do  not  mix. 
At  the  point  of  contact  note  the  various  colored  rings,  green, 
blue,  violet,  red  and  reddish-yellow.  Repeat  this  test  with 
different  dilutions  of  bile  and  observe  its  delicacy. 

(b)  Rosenbach's  Modification  of  Gmelin's  Test. — Filter  5 
c.c.  of  diluted  hile  through  a  small  filter  paper.  Introduce  a 
drop  of  concentrated  nitric  acid  into  the  cone  of  the  paper  and 
note  the  succession  of  colors  as  given  in  Gmelin's  test. 

(c)  Ilitppcrt's  Reaction. — Thoroughly  shake  equal  volumes 
of  undiluted  bile  and  milk  of  lime  in  a  test-tube.  The  pig- 
ments unite  with  the  calcium  and  are  precipitated.  Filter  off 
the  precipitate,  wash  it  with  water  and  transfer  to  a  small 
beaker.  Add  alcohol  acidified  slightly  with  hydrochloric  acid 
and  warm  upon  a  water-bath  until  the  solution  becomes  col- 
ored an  emerald  green. 

(d)  Hainmarstcn's  Reaction. — To  about  5  c.c.  of  Hammar- 
sten's  reagent1  in  a  small  evaporating  dish  add  a  few  drops  of 
diluted  bile.  A  green  color  is  produced.  If  more  of  the  rea- 
gent is  now  added  the  play  of  colors  as  observed  in  Gmelin's 
test  may  be  obtained. 

Hammarsten's  reagent  is  made  by  mixing  1  volume  of  25  per  cen!  nitric 
acid  and  ig  volumes  of  2$  per  cent  hydrochloric  acid  and  then  adding  I 
volume  of  this  acid  mixture  to  4  volumes  of  95  per  cent  alcohol. 


122  PHYSIOLOGICAL    CHEMISTRY. 

(e)  Smith's  Test. — To  2-3  c.c.  of  diluted  bile  in  a  test- 
tube  add  carefully  about  5  c.c.  of  dilute  tincture  of  iodine 
( 1 :  10)  so  that  the  fluids  do  not  mix.  A  play  of  colors,  green, 
blue  and  violet,  is  observed.  In  making  this  test  upon  the  urine 
ordinarily  only  the  green  color  is  observed.  Try  the  test  upon 
various  dilutions  of  bile  and  note  its  delicacy  as  compared 
with  that  of  Gmelin's  test.  Which  test  do  you  consider  the 
more  delicate  ? 

5.  Tests  for  Bile  Acids,  (a)  Pettenkofer's  Test. — To 
5  c.c.  of  diluted  bile  in  a  test-tube  add  5  drops  of  a  5  per 
cent  solution  of  saccharose.  Now  run  about  2-3  c.c.  of  con- 
centrated sulphuric  acid  carefully  down  the  side  of  the  tube 
and  note  the  red  ring  at  the  point  of  contact.  Upon  slightly 
agitating  the  contents  of  the  tube  the  whole  solution  gradu- 
ally assumes  a  reddish  color.  As  the  tube  becomes  warm,  it 
should  be  cooled  in  running  water  in  order  that  the  tempera- 
ture of  the  solution  may  not  rise  above  700  C. 

(b)  Mylius's  Modification  of  Pettenkofer's  Test.  To  ap- 
proximately 5  c.c.  of  diluted  bile  in  a  test-tube  add  3  drops  of 
a  very  dilute  (1  :  1,000)  aqueous  solution  of  furfurol, 

HC  — CH 

II       II 
HC      C  •  CHO. 
\/ 
0 

Now  run  about  2-3  c.c.  of  concentrated  sulphuric  acid  care- 
fully down  the  side  of  the  tube  and  note  the  red  ring  as  above. 
In  this  case  also,  upon  shaking  the  tube,  the  whole  solution  is 
colored  red.  Keep  the  temperature  of  the  solution  below  70  ° 
C.  as  before. 

( c)  Ncukomm's  Modification  of  Pettenkofer's  Test. — To 
a  few  drops  of  diluted  bile  in  an  evaporating  dish  add  a  trace 
of  a  dilute  saccharose  solution  and  one  or  more  drops  of  dilute 
sulphuric  acid.  Evaporate  on  a  water-bath  and  note  the 
development  of  a  violet  color  at  the  edge  of  the  evaporating 


BILE.  123 

mixture.     Discontinue  the  evaporation  as  soon  as  the  color  is 
observed. 

(d)  v.  Udrdnsky's  Test. — To  5  c.c.  of  diluted  bile  in  a  test- 
tube  add  3  4  drops  of  a  very  dilute  ( 1  :i,ooo)  aqueous  solution 
of  furfurol.  Place  the  thumb  over  the  top  of  the  tube  and 
shake  the  tube  until  a  thick  foam  is  formed.  By  means  O'f  a 
small  pipette  add  2—3  drops  of  concentrated  sulphuric  acid  to 
the  foam  and  note  the  dark  pink  coloration  produced. 

(e)  Hay's  Test. — Cool  about  10  c.c.  of  diluted  bile  in  a 
test-tube  to  \J°  C.  or  lower  and  sprinkle  a  little  finely  pulver- 
ized sulphur  upon  the  surface  of  the  fluid.  The  presence  of 
bile  acids  is  indicated  if  the  sulphur  sinks  to  the  bottom  of  the 
liquid,  the  rapidity  with  which  the  sulphur  sinks  depending 
upon  the  quantity  of  bile  acids  present  in  the  mixture.  The 
test  is  said  to  react  with  bile  acids  when  they  are  present  in 
the  proportion  1  :  120,000. 

Some  investigators  claim  that  it  is  impossible  to  differentiate 
between  bile  acids  and  bile  pigments  by  this  test. 

6.  Crystallization  of  Bile  Salts. — To  25  c.c.  of  undiluted 
bile  in  an  evaporating  dish  add  enough  animal  charcoal  to 
form  a  paste  and  evaporate  to  dryness  on  a  water-bath.  Re- 
move the  residue,  grind  it  in  a  mortar  and  transfer  it  to  a 
small  flask.  Add  about  50  c.c.  of  95  per  cent  alcohol  and  boil 
on  a  water-bath  for  20  minutes.  Filter,  and  add  ether  to  the 
filtrate  until  there  is  a  slight  permanent  cloudiness.  Cover  the 
vessel  and  stand  it  away  until  crystallization  is  complete. 
Examine  the  crystals  under  the  microscope  and  compare  them 
with  those  shown  in  Fig.  40,  page  118.  Try  one  of  the  tests 
for  bile  acids  upon  some  of  the  crystals. 


I24 


PHYSIOLOGICAL    CHEMISTRY. 


7.  Analysis  of  Biliary  Calculi. — Grind  the  calculus  in  a 
mortar  with  10  c.c.  of  ether.    Filter. 


Filtrate  I. 


Allow  to  evaporate  and  examine 
for  cholesterin  crystals  (Fig.  42, 
P-    125). 

(For  further  tests  see  experi- 
ment 8,  below.) 


Residue  I. 
(On  paper  and  in  mortar.) 

Treat  with  dilute  HC1  and  filter. 


Filtrate  II. 

Test  for  calcium,  phos- 
phates and  iron.  Evapo- 
rate remainder  of  filtrate 
to  dryness  in  porcelain 
crucible  and  ignite.  Dis- 
solve residue  in  dilute 
HC1  and  make  alkaline 
with  NH4OH.  Blue 
color  indicates  copper. 


Residue  II. 

(On  paper  and  in  mortar.) 
Wash    with    a    little    water.     Dry    the    filter 
paper. 

Treat  with  5  c.c.  chloroform  and  filter. 


Filtrate  III. 

Bilirubin. 
(Apply  test  for  bile 
pigments.) 


Residue  III. 

(On    paper    and    in 
mortar.) 


Treat  with  5  c.c.  of 
hot  alcohol. 


Biliverdin. 

8.  Tests  for  Cholesterin. 

(a)  Microscopical  Examination.  —  Examine  the  crystals 
under  the  microscope  and  compare  them  with  those  shown  in 
Fig.  42,  page  125. 

(b)  Iodine-Sulphuric  Acid  Test. — Place  a  few  crystals  of 
cholesterin  in  one  of  the  depressions  of  a  test-tablet  and  treat 
with  a  drop  of  concentrated  sulphuric  acid  and  a  drop  of  a 
very  dilute  solution  of  iodine.  A  play  of  colors  consisting  of 
violet,  blue,  green  and  red  results. 

(c)  The  Liebermann-Bnrchard  Test. — Dissolve  a  few  crys- 
tals of  cholesterin  in  2  c.c.  of  chloroform  in  a  dry  test-tube. 
Now  add  10  drops  of  acetic  anhydride  and  1-3  drops  of  con- 
centrated sulphuric  acid.  The  solution  becomes  red,  then  blue, 
and  finally  bluish-green  in  color. 


BILE. 


I  2 


(<!)  Salkoivski's  lest. — Dissolve  a  few  crystals  of  cho 
term  in  a  little  chloroform  and  add  an  equal  volume  of  con- 
centrated sulphuric  acid.     A  play  of  colors  from  bluish-red  to 
cherry-red  and  purple  is  noted  in  the  chloroform  while  the 
acid  assumes  a  marked  green  fluorescence. 

Fig.  42. 


Cholesterin. 


(  (• )  Sell  iff 's  Reaction. — To  a  little  cholesterin  in  an  evap<  >r- 
ating  dish  add  a  few  drops  of  a  mixture  of  3  volumes  of  con- 
centrated sulphuric  acid  and  1  volume  of  10  per  cent  ferric 
chloride.  Evaporate  to  dryness  over  a  low  flame  and  observe 
the  reddish-violet  residue  which  changes  to  a  bluish-violet. 

9.  Preparation  of  Taurin. — To  300  c.c.  of  bile  in  a  casse- 
role add  100  c.c.  of  hydrochloric  acid  and  heat  until  a  sticky 
mass  (dyslysin)  is  formed.  This  point  may  be  determined  by 
drawing  out  a  thread-like  portion  of  the  mass  by  means  of  a 
glass  rod,  and  if  it  solidifies  immediately  and  assumes  a  brittle 
character  we  may  conclude  that  all  the  taurocholic  and  glyco- 
cholic  acid  has  been  decomposed.  Decant  the  solution  and 
concentrate  it  to  a  small  volume  on  the  water-bath.  Filter  the 
hot  solution  to  remove  sodium  chloride  and  other  substances 
which  may  have  separated,  and  evaporate  the  filtrate  to  dry- 


126 


PHYSIOLOGICAL    CHEMISTRY. 


ness.  Dissolve  the  residue  in  5  per  cent  hydrochloric  acid 
and  precipitate  with  ten  volumes  of  95  per  cent  alcohol. 
Filter  off  the  taurin  and  recrystallize  it  from  hot  water. 
(Save  the  alcoholic  filtrate  for  the  preparation  of  glycocoll, 
page  127.)     Make  the  following  tests  upon  the  taurin  crystals : 

(a)  Examine  them  under  the  microscope  and  compare  with 
Fig.  43,  below. 

(b)  Heat  a  crystal  upon  platinum  foil.  The  taurin  at  first 
melts,  then  turns  brown  and  finally  carbonizes  as  the  tempera- 
ture is  raised.     Note  the  suffocating  odor.     What  is  it? 

(c)  Test  the  solubility  of  the  crystals  in  water  and  in 
alcohol. 

(d)  Grind  up  a  crystal  with  four  times  its  volume  of  dry 
sodium  carbonate  and  fuse  on  platinum  foil.  Cool  the  residue, 
transfer  it  to  a  test-tube  and  dissolve  it  in  water.     Add  a  little 


Taurin. 


dilute  sulphuric  acid  and  note  the  odor  of  hydrogen  sulphide. 
Hold  a  piece  of  filter  paper,  moistened  with  a  small  amount  of 
lead  acetate,  over  the  opening  of  the  test-tube  and  observe  the 
formation  of  lead  sulphide. 


BILK. 


127 


10.  Preparation  of  Glycocoll. — Concentrate  the  alcoholic 
filtrate  from  the  last  experiment   (9)   until  no  more  alcohol 

remains.  The  glyci  >e«  ill  is  present  here  in  the  form  of  an  hydro- 
chloride and  may  be  liberated  from  this  combination  by  the 
addition  of  freshly  precipitated  lead  hydroxide  or  by  lead 
hydroxide  solution.  Remove  the  lead  by  H2S.  Filter  and 
decolorize  the  filtrate  by  animal  charcoal.  Filter  again,  con- 
centrate the  filtrate  and  set  it  aside  for  crystallization.  Gly- 
cocoll  separates  as  colorless  crystals  |  Fig.  44,  below). 


Fig.  44. 


/7 


& 


' 


Glycocoll. 


11.  Synthesis  of  Hippuric  Acid. — To  some  of  the  glycocoll 
prepared  in  the  last  experiment  or  furnished  by  the  instructor, 
add  a  little  water,  about  1  c.c.  of  benzoyl  chloride  and  render 
alkaline  with  potassium  hydroxide  solution.  Stopper  the  tube 
and  shake  it  until  no  more  heat  is  evolved.  Now  render 
strongly  alkaline  with  potassium  hydroxide  and  shake  the 
mixture  until  no  odor  of  benzoyl  chloride  can  be  detected. 
Cool,  acidify  with  HC1,  add  an  equal  volume  of  petroleum 
ether  (ethyl  ether  may  be  substituted)  and  shake  thoroughly 
to  remove  the  benzoic  acid.  (Evaporate  this  solution  and  note 
the  crystals  of  benzoic  acid.     Compare  them  with  those  shown 


128  PHYSIOLOGICAL    CHEMISTRY. 

in  Fig.  94,  page  264. )  Decant  the  ethereal  solution  into  a  por- 
celain dish  and  extract  again  with  ether.  The  hippuric  acid 
remains  in  the  aqueous  solution.  Filter  it  off  and  wash  it  with 
a  small  amount  of  cold  water  while  still  on  the  filter.  Remove 
it  to  a  small  shallow  vessel,  dissolve  it  in  a  small  amount  of  hot 
water  and  set  it  aside  for  crystallization.  Examine  the  crystals 
microscopically  and  compare  them  with  those  in  Fig.  92, 
page  256. 

The  chemistry  of  the  synthesis  is  represented  thus : 

CH2-NH2  C0C1  OC-NH-CH2-COOH. 

/\  /\ 

+  11  =11 
COOH                \/  \/ 

Glycocoll.  Benzoyl  chloride.      Hippuric  acid. 


CHAPTER  IX. 
PUTREFACTION  PRODUCTS. 

The  putrefactive  processes  in  the  intestine  are  the  result  of 
the  action  of  bacteria  upon  the  proteid  material  present.  This 
bacterial  action  which  is  the  combined  effort  of  many  forms 
of  micro-organisms  is  confined  almost  exclusively  to  the  large 
intestine.  Some  of  the  products  of  the  putrefaction  of  proteids 
are  identical  with  those  formed  in  tryptic  digestion  although 
the  decomposition  of  the  proteid  material  is  much  more  ex- 
tensive when  subjected  to  putrefaction.  Some  of  the  more 
important  of  the  putrefaction  products  are  the  following: 
Indol,  skatol,  paracresol,  phenol,  para-ox  y  phenyl propionic  acid, 
para-oxyphenylacetic  acid,  volatile  fatty  acids,  hydrogen  sul- 
phide, methane,  methyl  mereaptan,  hydrogen,  and  carbon  diox- 
ide, beside  proteoses,  peptones,  ammonia  and  amino  acids.  Of 
these  the  indol,  skatol  and  phenol  appear  in  part  in  the  urine 
as  ethereal  sulphuric  acids,  whereas  the  oxyacids  mentioned 
pass  unchanged  into  the  urine.  The  potassium  indoxyl  sul- 
phate (page  130)  content  of  the  urine  is  a  rough  indicator  of 
the  extent  of  the  putrefaction  within  the  intestine. 

The  portion  of  the  indol  which  is  excreted  in  the  urine  is 
first  subjected  to  a  series  of  changes  within  the  organism  and 
is  subsequently  eliminated  as  indican.  These  changes  may  be 
represented  thus : 

/\ CH  /\ C(OH) 

I       I      II       +0  =  1      I      II 
\/\/CH  \/\/CB. 

NH  NH 

Jndol.  Indoxyl. 

/\ ('(OH)  /\ C(0-S03H) 

I      I      II  +HoS04H      I      II  +H20 

\/\/CH  \/\/CH 

NH  NH 

Indoxyl.  Indoxyl  sulphuric  acid. 

IO  129 


13°  PHYSIOLOGICAL    CHEMISTRY. 

In  the  presence  of  potassium  salts  the  indoxyl  sulphuric  acid 
is  then  transformed  into  potassium  indoxyl  sulphate  (or  indi- 
can), 

_C(0-S03K), 


\/\/CH 
NH 

and  eliminated  as  such  in  the  urine. 

Indican  may  be  decomposed  by  treatment  with  concentrated 
hydrochloric  acid  (see  tests  on  page  255)  into  sulphuric  acid 
and  indoxyl.  The  latter  body  may  then  be  oxidized  to  form 
indigo-blue  thus  : 

/\ C(OH)  /\ CO  OC /\ 

2  I       I      ||  +20=1       ||  ||       I  +2H20 

\/\/CH  \/\/C=C\/\/ 

NH  NH  NH       ' 

Indoxyl.  Indigo-blue. 

Skatol  is  likewise  changed  within  the  organism  and  elimi- 
nated in  the  form  of  a  chromogenic  substance. 

Experiments  on  Putrefaction  Products. 

In  many  courses  in  physiological  chemistry  the  instructors 
are  so  limited  for  time  that  no  extended  study  of  the  products 
of  putrefaction  can  very  well  be  attempted.  Under  such  con- 
ditions the  scheme  here  submitted  may  be  used  profitably  in  the 
way  of  a  demonstration.  Where  the  number  of  students  is 
not  too  great,  a  single  large  putrefaction  may  be  started,  and, 
after  the  initial  distillation,  both  the  resulting  distillate  and 
residue  may  be  distributed  to  the  members  of  the  class  for  in- 
dividual manipulation. 

Preparation  of  Putrefaction  Mixture. — Place  a  weighed 
mixture  of  coagulated  egg  albumin  and  ground  lean  meat  in 
a  flask  or  bottle  and  add  approximately  2  liters  of  water  for 
every  kilogram  of  proteid  used.  Sterilize  the  vessel  and  con- 
tents, inoculate  with  the  colon  bacillus  and  keep  at  400  C.  for 
two  or  three  weeks.     If  cultures  of  the  colon  bacillus  are  not 


PUT R 1. 1   \ci  [ON     PRODUCTS.  131 

available,  add  60  c.c.  of  a  cold  saturated  solution  of  sodium 

carh' mate  for  every  liter  of  water  previously  added  and  inocu- 
late with  s<mie  putrescent  material  (pancreas  or  feces).1  Mix 
the  putrefaction  mixture  very  thoroughly  by  shaking  and 
insert  a  cork  furnished  with  a  glass  tube  to  which  is  attached 
a  wash  bottle  containing  a  3  per  cent  solution  of  mercuric 
cyanide.2  This  device  is  for  the  purpose  of  collecting  the 
methyl  mecaptan,  a  gas  formed  during  the  process  1  >f  putrefac- 
tion. It  also  serves  to  diminish  the  odor  arising  from  the 
putrefying  material.  Place  the  putrefaction  mixture  at  40°  C. 
for  two  or  three  weeks  and  at  the  end  of  that  time  make  a 
separation  of  the  products  of  putrefaction  according  to  the 
following  directions: 

Subject  the  mixture  to  distillation  until  the  distillate  and 
residue  are  approximately  equal  in  volume. 

1  Putrefying  proteid  may  be  prepared  by  treating  10  grams  of  finely 
ground  lean  meat  with  ioo  c.c.  of  water  and  2  c.c.  of  a  saturated  solution 
of  sodium  carbonate  and  keeping  the  mixture  at  400  C.  for  twenty-four 
hours. 

2  Concentrated  sulphuric  acid  containing  a  small  amount  of  isatin  may 
be  used  as  a  substitute  for  mercuric  cyanide.  When  this  modification  is 
employed  it  is  necessary  to  use  calcium  chloride  tubes  to  exclude  moisture 
from  the  isatin  solution. 


132 


PHYSIOLOGICAL    CHEMISTRY, 


PART    I. 
MANIPULATION  OF  THE  DISTILLATE. 

Acidify   with   hydrochloric   acid   and   extract   with   ether. 


Ether  Extract  No.  i. 
Add  an  equal  volume  of  water, 
make  alkaline  with  potassium  hy- 
droxide  and    shake   thoroughly. 


Residue  No.  i. 

Allow  the  ether  to  volatilize. 
Evaporate  and  detect  ammonium 
chloride  crystals  (Fig.  45,  p.  133). 


Ether  Extract  No.  2. 
Evaporate  spontaneously.    Indol 
and  skatol  remain.    Try  proper  re- 
actions  (see  pages  136  and  137). 


Alkaline  Solution  No.  1. 

Acidify  with  hydrochloric  acid, 
add  sodium  carbonate  and  extract 
with  ether. 


Ether  Extract  No.  3. 
Evaporate.     Detect  phenol  and 
cresol  (paracresol).     See  p.  138. 


Alkaline  Solution  No.  2. 

Acidify  with  hydrochloric  acid, 
and   extract  with  ether. 


Ether  Extract  No.  4. 
Evaporate.     Volatile  fatty  acids 
remain. 


Final  Residue. 

(Discard.) 


DETAILED  DIRECTIONS  FOR  MAKING  THE 

SEPARATIONS  INDICATED  IN 

THE  SCHEME. 

Preliminary  Ether  Extraction. — This  extraction  may  be 
conveniently  conducted  in  a  separatory  funnel.  Mix  the  fluids 
for  extraction  in  the  ratio  of  two  volumes  of  ether  to  three 
volumes  of  the  distillate.  Shake  very  thoroughly  for  a  few 
moments,  then  draw  off  the  extracted  fluid  and  add  a  new  por- 
tion of  the  distillate.  Repeat  the  process  until  the  entire  dis- 
tillate  has   been   extracted.     Add   a   small   amount   of   fresh 


IT  |  KI.I    \CTION     PRODUCTS. 


133 


ether  at  each  extraction  to  replace  that  dissolved  by  the  water 
in  the  preceding  extraction. 

Residue  No.  1. — Unite  the  portions  of  the  distillate  extracted 
as    above    and    allow    the    ether    to    volatilize    spontaneously. 

Fig.  45- 


A  M  M  0  N  I  U  M    CH 1-OR I DE. 

Evaporate  until  crystallization  begins.  Examine  the  crystals 
under  the  microscope.  Ammonium  chloride  predominates. 
Explain  its  presence. 

Ether  Extract  No.  1. — Add  an  equal  volume  of  water,  ren- 
der the  mixture  alkaline  with  potassium  hydroxide  and  shake 
thoroughly  by  means  of  a  separator}-  funnel  as  before.  The 
volatile  fatty  acids,  contained  among  the  putrefaction  products, 
would  be  dissolved  by  the  alkaline  solution  (Xo.  1)  whereas 
any  indol  or  skatol  would  remain  in  the  ethereal  solution 
(No.  2). 

Alkaline  Solution  No.  1. — Acidify  with  hydrochloric  acid 
and  add  sodium  carbonate  solution  until  the  fluid  is  neutral  or 
slightly  acid  from  the  presence  of  carbonic  acid.  At  this  point 
a  portion  of  the  solution,  after  being  heated  for  a  few  moments, 
should  possess  an  alkaline  reaction  on  cooling.  Extract  the 
whole  mixture  with  ether  in  the  usual  way,  using  care  in  the 


134  PHYSIOLOGICAL    CHEMISTRY. 

manipulation  of  the  stop  cock  to  relieve  the  pressure  due  to 
the  evolution  of  carbon  dioxide.  The  ether  (Ether  Extract 
No.  3)  removes  any  phenol  or  cresol  which  may  be  present 
while  the  volatile  fatty  acids  will  remain  in  the  alkaline  solu- 
tion (No.  2)  as  alkali  salts. 

Ether  Extract  No.  2. — Drive  off  the  major  portion  of  the 
ether  at  a  low  temperature  on  a  water-bath  and  allow  the  resi- 
due to  evaporate  spontaneously.  Indol  and  skatol  should  be 
present  here.  Prove  the  presence  of  these  bodies.  For  tests 
for  indol  and  skatol  see  p.  — . 

Alkaline  Solution  No.  2. — Make  strongly  acid  with  hydro- 
chloric acid  and  extract  with  a  small  amount  of  ether,  using  a 
separatory  funnel.  As  carbon  dioxide  is  liberated  here,  care 
must  be  used  in  the  manipulation  of  the  stop  cock  of  the  funnel 
in  relieving"  the  pressure  within  the  vessel.  The  volatile  fatty 
acids  are  dissolved  by  the  ether  (Ether  Extract  No.  4). 

Ether  Extract  No.  5. — Evaporate  this  ethereal  solution  on  a 
water-bath.  The  oily  residue  contains  phenol  and  cresol.  The 
cresol  is  present  for  the  most  part  as  paracresol.  Add  some 
water  to  the  oily  residue  and  heat  it  in  a  flask.  Cool  and  prove 
the  presence  of  phenol  and  cresol.  For  tests  for  these  bodies 
see  page  138. 

Ether  Extract  No.  4. — Evaporate  on  a  water-bath.  The 
volatile  fatty  acids  remain  in  the  residge. 


PUTREFACTION  PRODUCTS. 


135 


PART    II. 
MANIPULATION  OF  THE  RESIDUE. 

Kaporate,  filter  and  extract  with  ether. 


Ether  Extract. 
Evaporate,  -extract    the    residue 
with  warm  water  and  filter. 


Aqueous  Solution. 
Evaporate  until  crystals  begin  to 
form.     Stand  in  a  cold  place  until 
crystallization  is  complete.     Filter. 


Crystalline  Deposit. 
Consists  of  a  mixture 
of  leiicin  and  tyrosin 
crystals.  (Figs.  23,  24 
and  104,  pages  68,  69  and 
326.) 


Filtrate  No.  1. 

Contains  proteose,  pep- 
tone, aromatic  acids  and 
tryptophan. 


Filtrate  No.  2. 
Contains   oxy acids  and 

skatol-carbonic  acid. 


Residue. 
Contains  non-volatile 

fatty  acids. 


DETAILED  DIRECTIONS  FOR  MAKING  THE 

SEPARATIONS  INDICATED  IN 

THE  SCHEME. 

Preliminary  Ether  Extraction. — This  extraction  may  be 
conducted  in  a  separatory  funnel.  In  order  to  make  a  satis- 
factory extraction  the  mixture  should  be  shaken  very  thor- 
oughly. Separate  the  ethereal  solution  from  the  aqueous  por- 
tion and  treat  them  according  to  the  directions  given  below. 

Ether  Extract. — Evaporate  this  solution  on  a  safety  water- 
bath  until  the  ether  has  been  entirely  removed.  Extract  the 
residue  with  warm  water  and  filter. 

Aqueous  Solution. — Evaporate  this  solution  until  crystalli- 
zation begins.  Stand  the  solution  in  a  cold  place  until  no 
more  crystals  form.  This  crystalline  mass  consists  of  impure 
leucin  and  tyrosin.     Filter  off  the  crystals. 


136  PHYSIOLOGICAL    CHEMISTRY. 

Crystalline  Deposit. — Examine  the  crystals  under  the  micro- 
scope and  compare  them  with  those  reproduced  in  Figs.  23, 
24  and  104,  pages  68,  69  and  326.  Do  the  forms  of  the 
crystals  of  leucin  and  tyrosin  resemble  those  previously  exam- 
ined? Make  a  separation  of  the  leucin  and  tyrosin  and  apply 
typical  tests  according  to  directions  given  on  pages  81  and  82. 

Filtrate  No.  1. — Make  a  test  for  tryptophan  with  bromine 
water  (see  page  no),  and,  also  with  the  Hopkins-Cole  rea- 
gent (see  page  45).  Use  the  remainder  of  the  filtrate  for 
the  separation  of  proteoses  and  peptones.  Make  the  separa- 
tion according  to  the  directions  given  on  page  5.9. 

Filtrate  No.  2. — This  solution  contains  para-oxyphenylacetic 
acid,  para-oxyphenylpropionic  acid  and  skatol-carbonic  acid. 
Prove  the  presence  of  these  bodies  by  appropriate  tests.  Tests 
for  oxyacids  and  skatol-carbonic  acid  are  given  on  page  138. 

TESTS  FOR  VARIOUS  PUTREFACTION 
PRODUCTS. 

Tests  for  Indol. 

1.  Herter's  Naphthaquinone  Reaction. —  (a)  To  a  dilute 
aqueous  solution  of  indol  (1  150,000)  add  one  drop  of  a  2  per 
cent  solution  of  naphthaquinone  sodium-monosulphonate.  No 
reaction  occurs.  Add  a  drop  of  a  10  per  cent  solution  of 
potassium  hydroxide  and  note  the  gradual  development  of  a 
blue  or  blue-green  color  which  fades  to  green  if  an  excess  of 
the  alkali  is  added.  Render  the  green  or  blue-green  solution 
acid  and  note  the  appearance  of  a  pink  color.  Heat  facilitates 
the  development  of  the  color  reaction. 

One  part  of  indol  in  one  million  parts  of  water  may  be  de- 
tected by  means  of  this  test  if  carefully  performed. 

(b)  If  the  alkali  be  added  to  the  indol  solution  before  the 
introduction  of  the  naphthaquinone  the  course  of  the  reaction 
is  different,  particularly  if  the  indol  solution  is  somewhat  more 
concentrated  than  that  mentioned  above  and  if  heat  is  used. 
Under  these  conditions  the  blue  indol  compound  ultimately 
forms  as  fine  acicular  crystals  which  rise  to  the  surface. 


PUTREI  \(   i  [ON    PRODUCT  S.  I  37 

If  we  do  not  wait  for  the  production  of  the  crystalline  liody 
but  as  soon  as  the  blue  color  forms,  shake  the  aqueous  solution 
with  chloroform,  the  blue  color  disappears  from  the  solution 
and  the  chloroform  assumes  a  pinkish-red  hue.  This  is  a 
distinguishing  feature  of  the  indol  reaction  and  facilitates  the 
differentiation  of  indol  from  other  bodies  which  yield  a 
similar  blue  color. 

2.  Cholera-red  Reaction. — To  a  little  of  the  residue  in  a 
test-tube  add  one-tenth  its  volume  of  a  0.02  per  cent  solution 
of  potassium  nitrite  and  mix  thoroughly.  Carefully  run  con- 
centrated sulphuric  acid  down  the  side  of  the  tube  so  that  it 
forms  a  layer  at  the  bottom.  Note  the  purple  color.  Neutra- 
lize with  potassium  hydroxide  and  observe  the  production  of 
a  bluish-green  color. 

3.  Legal's  Reaction. — To  a  small  amount  of  the  residue  in 
a  test-tube  add  a  few  drops  of  a  freshly  prepared  solution  of 
sodium  nitroprusside,  Na2Fe(CN)5NO  +  2ILO.  Render 
alkaline  with  potassium  hydroxide  and  note  the  production  of 
a  violet  color.  If  the  solution  is  now  acidified  with  glacial 
acetic  acid  the  voilet  is  transformed  into  a  blue. 

4.  Pine  Wood  Test. — Moisten  a  pine  splinter  with  con- 
centrated hydrochloric  acid  and  insert  it  into  the  residue.  The 
wood  assumes  a  cherry-red  color. 

5.  Nitroso-indol  Nitrate  Test. — Acidify  some  of  the  resi- 
due with  nitric  acid,  add  a  few  drops  of  a  potassium  nitrite 
solution  and  note  the  production  of  a  red  precipitate  of  nitroso- 
indol  nitrate.  If  the  residue  contains  but  little  indol  simply  a 
red  coloration  will  result.  Compare  this  result  with  the  result 
of  the  similar  test  on  skatol. 

Tests  for  Skatol. 

1.  Herter's  Naphthaquinone  Reaction. — The  same  pro- 
cedure may  be  used  here  as  in  the  similar  test  under  indol, 
page  136.  The  distinctive  feature  of  dilute  solutions  of  skatol 
when  treated  with  the  naphthaquinone  compound  is  that  they 
yield  a  violet  or  purple  instead  of  a  blue.     Concentrated  solu- 


138  PHYSIOLOGICAL    CHEMISTRY. 

tions  of  skatol  yield  the  blue  color  as  noted  with  indol.  This 
reaction  possesses  relatively  the  same  delicacy  as  the  indol 
reaction. 

2.  Color  Reaction  with  HC1. — Acidify  some  of  the  resi- 
due with  concentrated  hydrochloric  acid.  Note  the  production 
of  a  violet  color. 

3.  Acidify  some  of  the  residue  with  nitric  acid  and  add  a 
few  drops  of  a  potassium  nitrite  solution.  Note  the  white 
turbidity.  Compare  this  result  with  the  result  of  the  similar 
test  on  indol. 

Tests  for  Phenol  and  Cresol. 

1.  Color  Test. — Test  a  little  of  the  solution  with  Millon's 
reagent.  A  red  color  results.  Compare  this  test  with  the  simi- 
lar one  under  Tyrosin  (see  page  82). 

2.  Ferric  Chloride  Test. — Add  a  few  drops  of  neutral 
ferric  chloride  solution  to  a  little  of  the  residual  fluid.  A  dirty 
bluish-gray  color  is  formed. 

3.  Formation  of  Bromine  Compounds. — Add  some  bro- 
mine water  to  a  little  of  the  fluid  under  examination.  Note 
the  crvstalline  precipitate  of  tribromphenol  and  tribromcresol. 

Tests  for  Oxyacids. 

1.  Color  Test. — Test  a  little  of  the  solution  with  Millon's 
reagent.     A  red  color  results. 

2.  Bromine  Water  Test. — Add  a  few  drops  of  bromine 

water   to   some   of   the   filtrate.      A  turbidity   or   precipitate 

is  observed. 

Test  for  Skatol-carbonic  Acid. 

Ferric  Chloride  Test. — Acidify  some  of  the  filtrate  with 

hydrochloric  acid,  add  a  few  drops  of  ferric  chloride  solution 

and    heat.     Compare    the    end-reaction    with    that    given    by 

phenol. 


CHAPTER    X. 
FECES. 

The  feces  is  the  residual  mass  of  material  remaining  in  the 
intestine  after  the  full  and  complete  exercise  of  the  digestive 
and  absorptive  functions  and  is  ultimately  expelled  from  the 
body  through  the  rectum.  The  amount  of  this  fecal  discharge 
varies  with  the  individual  and  the  diet.  Upon  an  ordinary 
mixed  diet  the  daily  excretion  by  an  adult  male  will  aggregate 
1 10-170  grams  with  a  solid  content  ranging  between  25  and 
45  grams ;  the   fecal  discharge  of  such  an  individual  upon  a 

Fig.  46. 


Microscopical  Coxstitlknts  of  Feces.     ( v.  Jaksch.) 

a,  Muscle  fibers  ;  b,  connective  tissue ;  c,  epithelium  ;  d,  leucocytes ;  e,  spiral 
cells ;  /,  g,  h,  i,  various  vegetable  cells ;  k,  "  triple  phosphate "  crystals ;  /. 
woody  vegetable  cells ;  the  whole  interspersed  with  innumerable  micro- 
organisms of  various  kinds. 


vegetable  diet  will  be  much  greater  and  may  even  be  a* 
great  as  350  grams  and  possess  a  solid  content  of  75  grams. 
The  variation  in  the  normal  daily  output  being  so  great  ren- 
ders this  factor  of  very  little  value  for  diagnostic  purposes, 
except  where  the  composition  of  the  diet  is  accurately  known. 

•39 


140 


PHYSIOLOGICAL    CHEMISTRY. 


Lesions  of  the  digestive  tract,  a  defective  absorptive  function 
or  increased  peristalsis  as  well  as  an  admixture  of  mucus,  pus, 
blood  and  pathological  products  of  the  intestinal  wall  may- 
cause  the  total  amount  of  excrement  to  be  markedly  increased. 
The  fecal  pigment  of  the  normal  adult  is  hydrobilirubin 
(urobilin  or  stercobilin).  Neither  bilirubin  nor  biliverdin 
occurs  normally  in  the  fecal  discharge  of  adults,  although  the 
former  may  be  detected  in  the  excrement  of  nursing  infants. 
The  most  important  factor,  however,  in  determining  the  color 
of  the  fecal  discharge  is  the  diet.     A  mixed  diet  for  instance 

produces  stools  which  vary 
in  color  from  light  to  dark 
brown,  an  exclusive  meat 
diet  gives  rise  to  a  brown- 
ish-black stool,  whereas  the 
stool  resulting  from  a  milk- 
diet  is  invariably  light  col- 
ored. Certain  pigmented 
foods  such  as  the  chloro- 
phyllic  vegetables,  and  var- 
ious varieties  of  berries,  each 


Fig.  47- 


H^matoidin    Crystals    from    Acholic 

Stools.      (v.   Jaksch.) 
Color  of  crystals  same  as  the  color  of 
those  in  Fig.  41,  p.  119. 


afford  stools  having  a  char- 


acteristic color.  Certain 
drugs  ?ct  in  a  similar  way 
to  color  the  fecal  discharge.  This  is  well  illustrated  by  the 
occurrence  of  green  stools  following  the  use  of  calomel  and 
of  black  stools  after  bismuth  ingestion.  The  green  color  of 
the  calomel  stool  is  generally  believed  to  be  due  to  biliverdin. 
v.  Jaksch  however  claims  to  have  proven  this  view  to  be  in- 
correct since  he  was  able  to  detect  hydrobilirubin  (or  urobilin) 
but  no  biliverdin  in  stools  after  the  administration  of  calomel. 
The  bismuth  stool  derives  its  color  from  the  black  sulphide 
which  is  formed  from  the  subnitrate  of  bismuth.  In  cases 
of  biliary  obstruction  the  grayish-white  acholic  stool  is  formed. 
Under  normal  conditions  the  odor  of  feces  is  due  to  skatol 
and    indol,    two   bodies    formed    in   the   course    of   putrefac- 


I  ECES.  141 

tive  processes  occurring  within  the  intestine  (sec  page  129). 
Such  bodies  as  methane,  methyl  mercaptan  and  hydrogen  sul- 
phide may  also  add  to  the  disagreeable  character  of  the  odor. 
The  intensity  of  the  odor  depends  to  a  large  degree  upon  the 
character  of  the  diet,  being  very  marked  in  stools  from  a  meat 
diet,  much  less  marked  in  stools  from  a  vegetable  diet  and  fre- 
quently hardly  detectable  in  stools  from  a  milk  diet.  Thus 
the  stool  of  the  infant  is  ordinarily  nearly  odorless  and  any 
decided  odor  may  generally  be  readily  traced  to  some  patho- 
logical source. 

A  neutral  reaction  ordinarily  predominates  in  normal  stools 
although  slightly  alkaline  or  even  acid  stools  are  met  with. 
The  acid  reaction  is  encountered  much  less  frequently  than  the 
alkaline  and  then  commonly  only  following  a  vegetable  diet. 

The  form  and  consistency  of  the  stool  is  dependent,  in  large 
measure,  upon  the  nature  of  the  diet  and  particularly  upon 
the  quantity  of  water  ingested.      Under  nor- 
Fig.  48.  mai  con(]itions  the  consistency  may  vary  from 

a  thin,  pasty  discharge  to  a  firmly  formed 
stool.  Stools  which  are  exceedingly  thin 
and  watery  ordinarily  have  a  pathological 
significance.  In  general  the  feces  of  the  car- 
nivorous animals  is  of  a  firmer  consistency 
than  that  of  the  herbivora. 

Charcot-Leyden 

Crystals.  Among-  the  macroscopical  constituents  of 

the  feces  may  be  mentioned  the  following: 
Intestinal  parasites,  undigested  food  particles,  gall  stones, 
pathological  products  of  the  intestinal  wall,  enteroliths,  intes- 
tinal sand  and  objects  which  have  been  accidentally  swallowed. 
The  fecal  constituents  which  at  various  times  and  under 
different  conditions  may  be  detected  by  the  use  of  the  micro- 
scope are  as  follows :  Constituents  derived  from  the  food, 
such  as  muscle  fibers,  connective  tissue  shreds,  starch  granules 
and  fat;  formed  elements  derived  from  the  intestinal  tract, 
such  as  epithelium,  erythrocytes  and  leucocytes;  mucus;  pus 
corpuscles;  parasites  and  bacteria.     In  addition  to  the  consti- 


I42  PHYSIOLOGICAL    CHEMISTRY. 

tuents  named,  the  following  crystalline  deposits  may  be  de- 
tected: Cholestcrin,  fatty  acid,  fat,  bismuth  sulphide,  hcema- 
toidin,  "  triple  phosphate;''  Charcot-Leydcn  crystals  and  the 
oxalate,  carbonate,  phosphate,  sulphate  and  lactate  of  calcium. 

The  detection  of  minute  quantities  of  blood  in  the  feces 
("occult  blood")  has  recently  become  a  recognized  aid  to  a 
correct  diagnosis  of  certain  disorders.  In  these  instances  the 
hemorrhage  is  ordinarily  so  slight  that  the  identification  by 
means  of  macroscopical  characteristics  as  well  as  the  micro- 
scopical identification  through  the  detection  of  erythrocytes 
are  both  unsatisfactory  in  their  results.  Of  the  tests  given 
for  the  detection  of  "  occult  blood  "  the  aloin-turpentine  test 
(page  144)  is  probably  the  most  satisfactory.  Since  "occult 
blood  "  occurs  with  considerable  regularity  and  frequency  in 
gastrointestinal  cancer  and  in  gastric  and  duodenal  ulcer,  its 
detection  in  the  feces  is  of  especial  value  as  an  aid  to  a  correct 
diagnosis  of  these  disorders. 

For  diagnostic  purposes  the  macroscopical  and  microscopical 
examinations  of  the  feces  ordinarily  yield  much  more  satis- 
factory data  than  are  secured  from  its  chemical  examination. 

Experiments  on  Feces. 

1.  Macroscopical  Examination. — If  the  stool  is  watery 
pour  it  into  a  shallow  dish  and  examincdirectly.  If  it  is  firm 
or  pasty  it  should  be  treated  with  water  and  carefully  stirred 
before  the  examination  for  macroscopical  constituents  is 
attempted. 

The  macroscopical  constituents  may  be  collected  very  satis- 
factorily by  means  of  a  Boas  sieve  (Fig.  49,  page  143).  This 
sieve  is  constructed  of  two  easily  detachable  hemispheres  which 
are  held  together  by  means  of  a  bayonet  catch.  In  using  the 
apparatus  the  feces  is  spread  out  upon  a  very  fine  sieve  con- 
tained in  the  lower  hemisphere  and  a  stream  of  water  is  allowed 
to  play  upon  it  through  the  medium  of  an  opening  in  the 
upper  hemisphere.  The  apparatus  is  provided  with  an  orifice 
in   the  upper  hemisphere  through   which   the    feces   may  be 


I  KCKS. 


'    [3 


Fig.  -i" 


stirred  by  means  of  a  glass  rod  during  the  washing  proo 
After   [5—30  minutes  washing  nothing  but  the  coarse   Fecal 
constituents  remain  upon  the  sieve. 

2.  Microscopical  Examination. — Watery  stools  should  he 
placed  in  a  shallow  dish,  thoroughly  mixed  and  a  small  amount 
removed  to  a  slide  for  examination.  Stools 
of  a  firm  or  pasty  consistency  should  he 
rubbed  up  in  a  mortar  with  water  and  a 
small  portion  of  the  resulting-  mixture  trans- 
ferred to  a  slide  for  examination.  In  nor- 
mal feces  look  for  food  particles,  bacteria 
and  crystalline  bodies.  In  pathological 
stools,  in  addition  to  these  substances,  look 
for  animal  parasites  and  pathological  prod- 
ucts of  the  intestinal  wall. 

3.  Reaction. — Thoroughly  mix  the  feces 
and  apply  moist  red  and  blue  litmus  papers 
to  the  surface.  If  the  stool  is  hard  it  should 
be  mixed  with  water  before  the  reaction 
is  taken.  Examine  the  stool  as  soon  after 
defecation  as  is  convenient,  since  the  reac- 
tion may  change  very  rapidly.  The  reaction  of  the  normal 
stools  of  adult  man  is  ordinarily  neutral  or  faintly  alkaline 
to  litmus,  hut  seldom  acid.  Infants'  stools  are  generally  acid 
in  reaction. 

4.  Starch. — If  any  imperfectly  cooked  starch-containing 
food  has  been  ingested  it  will  be  possible  to  detect  starch 
granules  by  a  microscopical  examination  of  the  feces.  If  the 
granules  are  not  detected  by  a  microscopical  examination,  the 
feces  should  be  placed  in  an  evaporating  dish  or  casserole  and 
boiled  with  water  for  a  few  minutes.  Filter  and  test  the  filtrate 
by  the  iodine  test  in  the  usual  way  (see  page  24). 

5.  Cholesterin  and  Fat. — Extract  the  dry  feces  with  ether 
in  a  Soxhlet  apparatus  (see  Chapter  XXII).  If  this  apparatus 
is  not  available  transfer  the  dry  feces  to  a  tlask.  add  ether  and 
shake   frequently   for  a   few  hours.     Filter  and  remove  the 


Boas'    Sieve. 


144  PHYSIOLOGICAL    CHEMISTRY. 

ether  by  evaporation.  The  residue  contains  cholesterin  and 
the  mixed  fats  of  the  feces.  For  every  gram  of  fat  add 
about  iy2  grams  of  solid  potassium  hydroxide  and  25  c.c.  of 
95  per  cent  alcohol  and  boil  in  a  flask  on  a  water-bath  for  one- 
half  hour  maintaining  the  volume  of  alcohol  constant.  This 
alcoholic-potash  has  saponified  the  mixed  fats  and  we  now  have 
a  mixture  of  soaps  and  cholesterin.  Add  sodium  chloride,  in 
substance,  to  the  mixture  and  extract  with  ether  to  dissolve  out 
the  cholesterin.  Remove  the  ether  by  evaporation  and  ex- 
amine the  residue  microscopically  for  cholesterin  crystals. 
Try  any  of  the  other  tests  for  cholesterin  as  given  on  page  124. 

6.  Blood. — Undecomposed  blood  may  be  detected  macro- 
scopically.  If  uncertain,  look  for  erythrocytes  under  the 
microscope,  and  spectroscopically  for  the  spectrum  of  oxyhe- 
moglobin (see  Absorption  Spectra,  Plate  I). 

In  case  the  blood  has  been  altered  or  is  present  in  minute 
amount  ("occult  blood"),  and  cannot  be  detected  by  the 
means  mentioned  above,  the  following  tests  may  be  tried : 

(a)  Aloin-Turpentine  Test. — Mix  the  stool  very  thoroughly 
and  take  about  5  grams  of  the  mixture  for  the  test.  Reduce 
this  sample  to  a  semi-fluid  mass  by  means  of  distilled  water 
and  extract  very  thoroughly  with  an  equal  volume  of  ether 
to  remove  any  fat  which  may  be  present.  Now  treat  the  ex- 
tracted feces  with  one-third  its  volume,  of  glacial  acetic  acid 
and  10  c.c.  of  ether  and  extract  very  thoroughly  as  before. 
The  acid-ether  extract  will  rise  to  the  top  and  may  be  removed. 

Introduce  2-3  c.c.  of  this  acid-ether  solution  into  a  test-tube, 
add  an  equal  volume  of  a  dilute  solution  of  aloin  in  70  per 
cent  alcohol  and  2-3  c.c.  of  ozonized  turpentine  and  shake 
the  tube  gently.  If  blood  is  present  the  entire  volume  of  fluid 
ordinarily  becomes  pink  and  finally  cherry  red.  In  some  in- 
stances the  color  will  be  limited  to  the  aloin  solution  which 
sinks  to  the  bottom.  This  color  reaction  should  occur  within 
fifteen  minutes  in  order  to  indicate  a  positive  test  for  blood, 
since  the  aloin  will  turn  red  of  itself  if  allowed  to  stand  for  a 
longer  period.    The  color  is  ordinarily  light  yellow  in  a  nega- 


FECES.  I  }5 

tive  test.  Hydrogen  peroxide  is  not  a  satisfactory  substitute 
for  turpentine  in  this  test. 

{  b)  Weber's  Guaiac , Test. — Mix  a  little  feces  with  30  per 
cent  acetic  acid  to  form  a  fluid  mass.  Transfer  to  a  test-tube 
and  extract  with  ether.  If  blood  is  present  the  ether  will 
assume  a  brownish-red  color.  Filter  off  the  ether  extract  and, 
to  a  portion  of  the  filtrate,  add  an  alcoholic  solution  of  guaiac 
(strength  about  1  :6b),1  drop  by  drop,  until  the  fluid  becomes 
turbid.  Xow  add  hydrogen  peroxide  or  old  turpentine.  In 
the  presence  of  blood  a  blue  color  is  produced  (see  page  158). 

(c)  Acid-Hccmatin. — Examine  some  of  the  ethereal  extract 
from  the  last  experiment  (b)  spectroscopically.  Note  the 
typical  spectrum  of  acid-haematin  (see  Absorption  Spectra, 
Plate  II). 

7.  Hydrobilirubin. — Rub  up  a  small  amount  of  feces  in  a 
mortar  with  a  concentrated  aqueous  solution  of  mercuric 
chloride.  Transfer  to  a  shallow  flat-bottomed- dish  and  allow 
to  stand  several  hours.  The  presence  of  hydrobilirubin  will  be 
indicated  by  a  deep  red  color  imparted  to  the  feces.  This  red 
color  is  due  to  the  formation  of  hydrobilirubin-mercury.  If 
unaltered  bilirubin  is  present  the  feces  will  be  green  in  color. 

Another  method  for  the  detection  of  hydrobilirubin  is  the 
following:  Treat  the  dry  feces  with  absolute  alcohol  acidified 
with  sulphuric  acid  and  shake  thoroughly.  The  acidified  alco- 
hol extracts  the  pigment  and  assumes  a  reddish  color.  Ex- 
amine a  little  of  this  fluid  spectroscopically  and  note  the  typical 
spectrum  of  hydrobilirubin  (Absorption  Spectra  Plate  II). 

8.  Bilirubin,  (a)  Gmeliris  Test. — Place  a  few  drops  of 
concentrated  nitric  acid  in  an  evaporating  dish  or  on  a  porce- 
lain test-tablet  and  allow  a  few  drops  of  feces  and  water  to 
mix  with  it.  The  usual  play  of  colors  of  Gmeliirs  test  is  pro- 
duced, i.  e.j  green,  blue,  violet,  red  and  yellow.  If  so  desired, 
this  test  may  be  executed  on  a  slide  and  observed  under  the 
microscope. 

1  Buckmaster    advises    the    use    of    an    alcoholic    solution    of    guaiaconic 
acid  instead  of  an  alcoholic  solution  of  guaiac  resin. 


I46  PHYSIOLOGICAL    CHEMISTRY. 

(b)  Hupperfs  Test. — Treat  the  feces  with  water  to  form 
a  semi-fluid  mass,  add  an  equal  amount  of  milk  of  lime,  shake 
thoroughly  and  filter.  Wash  the  precipitate  with  water,  then 
transfer  both  the  paper  and  the  precipitate  to  a  small  beaker 
or  flask,  add  a  small  amount  of  95  per  cent  alcohol  acidified 
slightly  with  sulphuric  acid  and  heat  to  boiling  on  a  water- 
bath.  The  presence  of  bilirubin  is  indicated  by  the  alcohol 
assuming  a  green  color. 

9.  Bile  Acids. — Extract  a  small  amount  of  feces  with  alco- 
hol and  filter.  Evaporate  the  filtrate  on  a  water-bath  to  drive 
off  the  alcohol  and  dissolve  the  residue  in  water  made  slightly 
alkaline  with  potassium  hydroxide.  Upon  this  aqueous  solu- 
tion .try  any  of  the  tests  for  bile  acids  given  on  page  122. 

10.  Caseinogen. — Extract  the  fresh  feces  first  with  a  dilute 
solution  of  sodium  chloride,  and  later  with  water  acidified  with 
dilute  acetic  acid,  to  remove  soluble  proteids.  Now  extract 
the  feces  with  0.5  per  cent  sodium  carbonate  and  filter.  Add 
dilute  acetic  acid  to  the  filtrate  to  precipitate  the  caseinogen, 
being  careful  not  to  add  an  excess  of  the  reagent  as  the  casein- 
ogen would  dissolve.  Filter  off  the  caseinogen  and  test  it 
according  to  directions  given  on  page  192.  Caseinogen  is 
found  principally  in  the  feces  of  children  who  have  been  fed  a 
milk  diet.  Mucin  would  also  be  extracted  by  the  dilute  alkali, 
if  present  in  the  feces.  What  test  cotild  you  make  on  the 
newly  precipitated  body  to  differentiate  between  mucin  and 
caseinogen  ? 

11.  Nucleoproteid. — Mix  the  stool  thoroughly  with  water, 
transfer  to  a  flask,  and  add  an  equal  amount  of  saturated  lime 
water.  Shake  frequently  for  a  few  hours,  filter,  and  precipi- 
tate the  nucleoproteid  with  acetic  acid.  Filter  off  this  precipi- 
tate and  test  it  as  follows : 

(a)  Phosphorus. — Test  for  phosphorus  by  fusion  (see 
page  223). 

(b)  Solubility. — Try  the  solubility  in  the  ordinary  solvents. 

(c)  Proteid  Color  Test. — Try  any  of  the  proteid  color  tests. 
What  proof  have  you  that  the  above  body  was  not  mucin? 


FECES.  147 

What  other  test  can  you  use  to  differentiate  between  nucleo- 
proteid  and  mucin?' 

12.  Albumin  and  Globulin. — Extract  the  fresh  feces  with 
a  dilute  solution  of  sodium  chloride.  (The  preliminary  extract 
from  the  preparation  of  caseinogen,  page  146,  may  be  utilized 
here).  Filter,  and  saturate  a  portion  of  the  filtrate  with 
sodium  chloride  in  substance.  A  precipitate  signifies  globulin. 
Filter  off  the  precipitate  and  acidify  the  filtrate  slightly  with 
dilute  acetic  acid.  A  precipitate  at  this  point  signifies  albumin. 
Make  a  proteid  color  test  on  each  of  these  bodies. 

13.  Proteose  and  Peptone. — Heat  to  boiling  the  portion 
of  the  sodium  chloride  extract  not  used  in  the  last  experiment. 
Filter  off  the  coagulum,  if  any  forms.  Acidify  the  filtrate 
slightly  with  acetic  acid  and  saturate  with  sodium  chloride  in 
substance.  A  precipitate  here  indicates  proteose.  Filter  it 
off  and  test  it  according  to  directions  given  on  page  59.  Test 
the  filtrate  for  peptone  by  the  biuret  test. 

14.  Inorganic  Constituents. — Prepare  a  dilute  aqueous 
solution  of  dry  feces  and  decolorize  it  by  means  of  purified 
animal  charcoal.  Make  the  following  tests  upon  the  clear 
solution : 

(a)  Chlorides. — Acidify  with  nitric  acid  and  add  silver 
nitrate. 

(b)  Phosphates. — Acidify  with  nitric  acid,  add  molybdic 
solution  and  warm  gently. 

(c)  Sulphates. — Acidify  with  hydrochloric  acid,  add  barium 
chloride  and  warm. 


CHAPTER    XI. 
BLOOD. 

Blood  is  composed  of  three  types  of  form-elements  (ery- 
throcytes or  red  blood  corpuscles,  leucocytes  or  white  blood 
corpuscles  and  blood  plates  or  plaques)  held  in  suspension  in 
a  fluid  called  blood  plasma.  These  form-elements  compose 
about  60  per  cent  of  the  blood,  by  weight.  Ordinarily  blood 
is  a  dark  red,  opaque  fluid  due  to  the  presence  of  the  red  blood 
corpuscles,  but  through  the  action  of  certain  substances  such 
as  water,  ether  or  chloroform  it  may  be  rendered  transparent. 
Blood  so  altered  is  said  to  be  laked.  The  laking  process  is 
simply  a  liberation  of  the  haemoglobin  from  the  stroma  of  the 
red  blood  corpuscle.  Normal  blood  is  alkaline  in  reaction1  to 
litmus,  the  alkalinity  being  due  principally  to  sodium  carbonate. 
The  specific  gravity  of  the  blood  of  adults  ordinarily  varies 
between  1.045  an(^  l-°7S-  It  varies  somewhat  with  the  sex, 
the  blood  of  males  having  a  rather  higher  specific  gravity  than 
that  of  females.  Under  pathological  conditions  also  the 
density  of  the  blood  may  be  very  greatly  altered.  The  freez- 
ing-point (A)  of  normal  blood  is  about  — 0.560  C.  Varia- 
tions between  — 0.51  °  and  — 0.62 °  C.  may  be  due  entirely,  to 
dietary  conditions,  but  if  any  marked  variation  is  noted  it  can, 
in  most  cases,  be  traced  to  a  disordered  kidney  function.  The 
total  amount  of  blood  in  the  body  has  been  variously  estimated 
at  from  one-twelfth  to  one-fourteenth  of  the  body  weight. 
Perhaps  1/13.5  is  the  most  satisfactory  figure. 

Among  the  most  important  constituents  of  blood  plasma  are 
the  four  proteid  bodies,  fibrinogen,  nucleoproteid,  scrum 
globulin  (euglobulin  and  pseudo-globulin)  and  serum  albumin. 
Plasma  contains  about  8.2  per  cent  of  solids  of  which  the 

1  Recently  it  has  been  shown  by  physico-chemical  methods  that  the  blood 
is  in   reality  neutral   in   reaction. 

148 


BLOOD.  I  f9 

proteid  constituents  named  above  constitute  approximately  84 

per  cent  and  the  inorganic  constituents  (mainly  chlorides, 
phosphates  and  carbonates)  approximately  10  per  cent. 
Among  the  inorganic  constituents  sodium  chloride  predomi- 
nates.    To  prevent  coagulation,  blood  plasma   is  ordinarily 

studied  in  the  form  of  an  oxalated  or  suited  plasma.  The 
former  may  he  obtained  by  allowing  the  blood  to  flow  from  an 
opened  artery  into  an  equal  volume  of  0.2  per  cent  ammonium 
oxalate  solution,  whereas  in  the  preparation  of  a  sailed  plasma 
10  per  cent  sodium  chloride  solution  may  be  used  as  the  dilut- 
ing tin  id. 

Fibrinogen  is  perhaps  the  most  important  of  the  proteid 
constituents  of  the  plasma.  It  is  also  found  in  lymph  and  chyle 
as  well  as  in  certain  exudates  and  transudates.  Fibrinogen 
possesses  the  general  properties  of  the  globulins,  but  differs 
from  serum  globulin  in  being  precipitated  upon  half-saturation 
with  sodium  chloride.  In  the  process  of  coagulation  of  the 
blood  the  fibrinogen  is  transformed  into  fibrin.  This  fibrin 
is  one  of  the  principal  constituents  of  the  ordinary  blood  clot. 

The  nucleo-proteid  of  blood  possesses  many  of  the  charac- 
teristics of  serum  globulin.  In  common  with  this  body  it  is 
easily  soluble  in  sodium  chloride,  and  is  completely  precipi- 
tated from  its  solutions  upon  saturation  with  magnesium  sul- 
phate. It  is  much  less  soluble  in  dilute  acetic  acid  than  serum 
globulin  and  its  solutions  coagulate  at  65°-69°  C. 

The  body  formerly  called  serum  globulin  is  probably  not  an 
individual  substance.  Recent  investigations  seem  to  indicate 
that  it  may  be  resolved  into  two  individual  bodies  called 
euglobulin  and  pseudo globulin.  The  euglobulin  is  practically 
insoluble  in  water  and  may  be  precipitated  in  the  presence  of 
28-36  per  cent  of  saturated  ammonium  sulphate  solution.  The 
pseudoglobulin,  on  the  contrary,  is  soluble  in  water  and  is  only 
precipitated  by  ammonium  sulphate  in  the  presence  of  from 
36  to  44  per  cent  of  saturated  ammonium  sulphate  solution. 

In  common  with  serum  globulin  the  body  known  as  serum 
albumin  seems  also  to  consist  of  more  than  a  single  individual 


I50  PHYSIOLOGICAL    CHEMISTRY. 

substance.  The  so-called  serum  albumin  may  be  separated  into 
at  least  two  distinct  bodies,  one  capable  of  crystallization,  the 
other  an  amorphous  body.  The  solution  of  either  of  these 
bodies  in  water  gives  the  ordinary  albumin  reactions.  The 
coagulation  temperature  of  the  serum  albumin  mixture  as  it 
occurs  in  serum  or  plasma  varies  from  70 °  to  85  °  C.  according 
to  the  reaction  of  the  solution  and  its  content  of  inorganic 
material.  Serum  albumin  differs  from  Qgg  albumin  in  being 
more  laevorotatory,  in  being  rendered  less  insoluble  by  alcohol, 
and  in  the  fact  that  when  precipitated  by  hydrochloric  acid  it  is 
more  easily  soluble  in  an  excess  of  the  reagent. 

When  blood  coagulates  and  the  usual  clot  forms,  a  light  yel- 
lcw  fluid  exudes.  This  is  blood  serum.  It  differs  from  blood 
plasma  in  containing  a  large  amount  of  fibrin  ferment,  a  body 
of  great  importance  in  the  coagulation  of  the  blood,  and  also 
in  possessing  a  lower  proteid  content.  The  proteid  material 
present  in  plasma  and  not  found  in  serum  is  the  fibrinogen 
which  is  transformed  into  fibrin  in  the  process  of  coagulation 
and  removed.  The  specific  gravity  of  the  serum  of  human 
blood  varies  between  1.026  and  1.032. 

Beside  the  proteid  constituents  already  mentioned,  other 
bodies  which  are  found  in  both  the  plasma  and  serum  are  the 
following:  Sugar  (dextrose),  fat,  enzymes,  lecithin,  choles- 
terin  and  its  esters,  gases,  coloring-matter  (lutein  or  lipo- 
chrome)  and  mineral  substances.  In  addition  to  these  bodies 
the  following  substances  have  been  detected  in  normal  human 
blood:  Creatin,  carbamic  acid,  hippuric  acid,  paralactic  acid, 
area  and  uric  acid.  Some  of  the  pathological  constituents  of 
blood  are,  proteoses,  leucin,  tyrosin  and  other  amino  acids, 
biliary  constituents  and  purin  bodies. 

There  has  recently  been  considerable  controversy  regarding 
the  form  of  the  erythrocytes  or  red  blood  corpuscles  of  human 
blood.  It  is  claimed  by  some  investigators  that  the  cells  are 
bell-shaped  or  cup-shaped.  As  the  erythrocytes  occur  nor- 
mally in  the  circidation,  however,  they  are  probably  thin,  non- 
nucleated,   biconcave   discs.     When   examined   singly,    under 


PLATE    IV. 


Nokmal    Erythrocytes    and    Leucocytes. 


BLOOD.  151 

the  microscope,  they  possess  a  pale  greenish-yellow  color  (see 
Plate  IV,  opposite),  whereas  when  grouped  in  large  masses  a 
reddish  tint  is  noted. 

The  blood  of  most  mammals  contains  erythrocytes  similar 
in  form  to  those  of  human  blood.  In  the  blood  of  a  few 
mammals,  however,  such  as  the  llama  and  camel  as  well  as  in 
the  blood  of  birds,  fishes,  amphibians  and  reptiles  the  ery- 
throcytes are  ordinarily  more  or  less  elliptical,  biconvex  and 
possess  a  nucleus.  The  erythrocytes  vary  in  size  with  the 
different  animals.  The  average  diameter  of  the  erythrocytes 
of  blood  from  various  species  is  given  in  the  following  table  :* 

Elephant 3  fa ^  of  an  inch. 

Guinea-pig jj'ij  of  an  inch. 

Man JT3JJ  of  an  inch. 

Monkey 7 jjt  of  an  inch. 

Dog ttoVt  of  an  inch. 

Rat xfan  OI  an  inch. 

Rabbit jrfo  of  an  inch. 

Mouse g  yVr  of  an  inch. 

Lion 4"ttt  of  an  inch. 

Ox T$tv  °f  an  inch. 

Horse TjTT  of  an  inch. 

Pig T yjj  of  an  inch. 

Cat T jVj  of  an  inch. 

Sheep ■•  4  yV?  of  an  inch. 

Goat ^xV?  of  an  inch. 

Musk-deci ti1!;?  of  an  inch. 

The  erythrocytes  from  whatever  source  obtained,  consist 
essentially  of  two  parts,  the  stroma  or  protoplasmic  tissue  and 
its  enclosed  pigment,  Jiccmoglobin.  For  human  blood  the 
number  of  erythrocytes  present  in  the  fluid  as  obtained  from 
well-developed  males  in  good  physical  condition  is  about 
5,500,000  per  cubic  millimeter.2  The  normal  content  of  the 
blood  of  adult  females  is  from  4,000,000  to  4,500,000  per 
cubic  millimeter.     The  number  of  erythrocytes  varies  greatly 

1  Wormley's  Micro-Chemistry  of  Poisons,  second  edition,  p.  733. 

2  This  statement  is  based  upon  observations  made  upon  the  blood  of 
athletes  in  training.  It  is  generally  stated  in  text-books  that  the  blood 
of  males  contains  about  5,000,000  per  cubic  millimeter. 


152 


PHYSIOLOGICAL    CHEMISTRY. 
Fig.  50. 


^8?«*  * 


Oxyhemoglobin  Crystals  from  Blood  of  the  Guinea  Pig. 

Reproduced   from   a   micro-photograph   furnished  by   Prof.   E.    T.   Reichert   of 
the   University   of    Pennsylvania. 


Fig.  51. 


Oxyhemoglobin  Crystals  from  Blood  of  the  Rat. 

Reproduced   from   a   micro-photograph   furnished   by   Prof.    E.   T.   Reichert   of 
the  University   of   Pennsylvania. 


JU.OOD. 


'53 


Oxyhemoglobin  Crystals  from  Blood  of  the  Horse. 

Reproduced   from   a   micro-photograph    furnished   by   Prof.    E.   T.    Reichert    of 
the  University   of   Pennsylvania. 


Fig.  :3. 


ftd 


^ 


& 


Oxyhemoglobin  Crystals  from  Blood  of  the  Squirrel. 

Reproduced   from   a   micro-photograph    furnished  by    Prof.    E.   T.    Reichert   of 
the  University   of   Pennsylvania. 


x54 


PHYSIOLOGICAL    CHEMISTRY, 
Fig.  54. 


Oxyhemoglobin   Crystals  from  Blood  of  the  Dog. 

Reproduced   from   a  micro-photograph   furnished  by   Prof.   E.   T.    Reichert   of 
the  University   of   Pennsylvania. 


Fig.  55- 


Oxyhemoglobin   Crystals  from  Blood  of  the  Cat. 

Reproduced   from   a  micro-photograph   furnished   by   Prof.   E.   T.    Reichert   of 
the  University   of    Pennsylvania. 


BLOOD. 

Fig.  56. 


■D3 


Oxyhemoglobin   Crystals  from  Blood  of  thk  Nectlrus. 

Reproduced   from   a   micro-photograph   furnished  by    Prof.    E.   T.    Reichert    of 

the    University    of    Pennsylvania.1 

under  different  conditions.  For  instance  the  number  may  be 
increased  after  the  transfusion  of  blood  of  the  same  species 
of  animal;  by  residing  in  a  high  altitude;  or  as  a  result  of 
strenuous  physical  exercise  continued  over  a  short  period  of 
time.  An  increase  is  also  noted  in  starvation;  after  partaking 
of  food;  after  cold  or  hot  baths;  after  massage,  as  well  as 
after  the  administration  of  certain  drugs  and  accompanying 
certain  diseases  such  as  cholera,  diarrhoea,  dysentery  and  yel- 
low atrophy  of  the  liver.  A  decrease  in  the  number  occurs  in 
the  different  forms  of  anaemia.  The  number  has  been  known 
to  increase  to  7,040,000  per  cubic  millimeter  as  a  result  of 
physical  exercise,  while  11,000,000  per  cubic  millimeter  have 
been  noted  in  cases  of  polycythemia  and  increases  nearly  as 
great  in  cyanosis.  The  number  has  been  known  to  decrease 
to  500,000  per  cubic  millimeter  or  lower  in  pernicious  anaemia. 
Oxyhemoglobin  the  coloring  matter  of  the  blood  is  a  com- 

'The  micro-photographs  of  oxyhemoglobin  (see  pages  152-155)  and 
haemin  (see  page  164)  are  reproduced  through  the  courtesy  of  Profes- 
sors E.  T.  Reichert  and  Amos  P.  Brown,  of  the  University  of  Pennsyl- 
vania, who  are  investigating  the  crystalline  forms  of  biochemic  substances. 


is6 


PHYSIOLOGICAL    CHEMISTRY 


pound  proteid.  Through  treatment  with  hydrochloric  acid  it 
may  be  split  into  a  proteid  body  called  globin,  and  hcemo- 
chromogcn  an  iron-containing  pigment.  The  latter  body  is 
rapidly  transformed  into  hcematin  in  the  presence  of  oxygen 
and  this  in  turn  gives  place  to  hsematin-hydrochloride  or 
Juoniii  (  Figs.  58  and  59,  page  164).  The  pigment  of  arterial 
blood  is  for  the  most  part  loosely  combined  with  oxygen  and 
is  termed  oxyhemoglobin,  whereas  the  pigment  of  venous 
blood  is  principally  haemoglobin  (so-called  reduced  haemoglo- 
bin). Oxyhaemoglobin  is  the  oxygen-carrier  of  the  body  and 
belongs  to  the  class  of  bodies  known  as  respiratory  pigments. 
The  reduction  of  oxyhaemoglobin  to  form  haemoglobin  (so- 
called  reduced  haemoglobin)  occurs  in  the  capillaries.  Oxy- 
haemoglobin may  be  crystallized  and  a  specific  form  of  crystal 
obtained  from  the  blood  of  each  individual  species  (see  Figs. 
50  to  56,  pages  152  to  155).  This  fact  seems  to  indicate 
that  there  are  many  varieties  of  oxyhaemoglobin.  The  pig- 
ment is  held  within  the  stroma  of  the  erythrocyte.  The  fol 
lowing  bodies  may  be  derived  from  haemoglobin,  and  each 
possesses  a  specific  spectrum  which  serves  as  an  aid  in  its 
detection  and  identification :  Oxyhaemoglobin,  methaemoglo- 
bin,  carbon-monoxide  haemoglobin,  nitric-oxide  haemoglobin, 
haemochromogen,  haematin,  acid-haematin,  alkali-haematin  and 
haematoporphyrin  (see  Absorption  Spectra,  Plates  I  and  II). 
The  white  corpuscles  (or  leucocytes)  of  human  blood  differ 
from  the  red  corpuscles  (or  erythrocytes)  in  being  somewhat 
larger  in  size,  in  containing  at  least  a  single  nucleus  and  in 
possessing  amoeboid  movement  (see  Plate  IV,  opposite  page 
151).  They  are  typical  animal  cells  and  therefore  contain  the 
following  bodies  which  are  customarily  present  in  such  cells : 
Proteids,  fats,  carbohydrates,  lacithin,  cholcsterin,  inorganic 
salts  and  water.  The  normal  number  of  leucocytes  in  human 
blood  varies  between  5,000  and  10,000  per  cubic  millimeter. 
The  ratio  between  the  leucocytes  and  erythrocytes  is  about 
1  :  350-500.  A  leucocytosis  is  said  to  exist  when  the  number 
of  leucocytes  is  increased  for  any  reason.     Leucocytoses  may 


BLOOD.  !57 

be  divided  into  two  general  classes,  the  physiological  and  the 
pathological.  Under  the  physiological  form  would  be  classed 
those  leucocytoses  accompanying  pregnancy,  parturition  and 
digestion,  as  well  as  those  due  to  mechanical  and  thermal 
influences.  The  leucocytoses  spoken  of  as  pathological  are 
the  inflammatory,  infectious,  post-haemorrhagic,  toxic  and 
experimental  forms  as  well  as  the  type  of  leucocytosis  which 
accompanies  malignant  disease. 

The  blood  plates  (platelets  or  plaques)  arc  round  or  oval, 
colorless  discs  which  possess  a  diameter  about  one-third  as 
great  as  that  of  the  erythrocytes.  Upon  treatment  with  certain 
reagents,  c.  g.,  artificial  gastric  juice,  they  may  be  separated 
into  a  homogeneous,  non-refractive  portion  and  a  granular, 
refractive  portion.  The  blood  plates  are  probably  associated 
in  some  way  with  the  coagulation  of  the  blood.  This  relation- 
ship is  not  well  understood  at  present. 

The  processes  involved  in  the  coagulation  of  the  blood  are 
not  fully  understood.  Several  theories  have  been  advanced 
and  each  has  its  adherents.  The  theory  which  appears  to  be 
fully  as  firmly  founded  upon  experimental  evidence  as  any  is 
the  following-:  Blood  contains  a  zymogen  called  prothrombin 
which  combines  with  the  calcium  salts  present  to  form  an 
enzyme  known  as  thrombin  or  fibrin-ferment.  When  freshly 
drawn  blood  comes  in  contact  with  the  air  the  fibrin- ferment 
at  once  acts  upon  the  fibrinogen  present  and  gives  rise  to  the 
formation  of  fibrin.  This  fibrin  forms  in  shreds  throughout 
the  blood  mass  and,  holding  the  form  elements  of  the  blood 
within  its  meshes,  serves  to  produce  the  typical  blood  clot. 
The  fibrin  shreds  gradually  contract,  the  whole  clot  assumes 
a  jelly-like  appearance  and  the  yellowish  serum  exudes.  If, 
immediately  upon  the  withdrawal  of  blood  from  the  body,  the 
fluid  be  rapidly  stirred  or  thoroughly  "  whipped  "  with  a 
bundle  of  coarse  strings,  twigs  or  a  specially  constructed 
beater,  the  fibrin  shreds  will  not  form  in  a  network  through- 
out the  blood  mass  but  instead  will  cling  to  the  device  used 
in  beating.     In  this  way  the  fibrin  may  be  removed  and  the 


158  PHYSIOLOGICAL    CHEMISTRY. 

remaining  fluid  is  termed  defibrinatcd  blood.  The  above 
theory  of  the  coagulation  of  the  blood  may  be  stated  briefly 
as  follows : 

I.  Prothrombin  -f-  Calcium  Salts  =  Thrombin  (or  Fibrin- 
ferment). 

II.  Thrombin  (or  Fibrin-ferment)  +  Fibrinogen  =  Fibrin. 
Among  the  medico-legal  tests  for  blood  are  the  following: 

(1)  Microscopical  identification  of  the  erythrocytes,  (2)  spec- 
troscopic identification  of  blood  solutions,  (3)  the  guaiac  test, 
(4)  preparation  of  hsemin  crystals.  Of  these  four  tests  the 
last  named  is  generally  considered  to  be  the  most  satisfactory. 
It  gives  equally  reliable  results  with  fresh  blood  and  with  blood 
from  clots  or  stains  of  long  standing,  provided  the  latter  have 
not  been  exposed  to  a  high  temperature,  or  to  the  rays  of  the 
sun  for  a  long  period.  The  technique  of  the  test  is  simple 
(see  page  163)  and  the  formation  of  the  dark  brown  or 
chocolate  colored  crystals  of  haemin  (Figs.  58  and  59,  page 
164)  is  indisputable  proof  of  the  presence  of  blood  in  the 
fluid,  clot  or  stain  examined.  The  weak  point  of  the  test, 
medico-legally,  lies  in  the  fact  that  it  does  not  differentiate 
between  human  blood  and  that  of  certain  other  species  of 
animal. 

The  guaiac  test  (see  page  163),  although  generally  con- 
sidered less  accurate  than  the  hsemin  test,  is  really  a  more  deli- 
cate test  than  the  hsemin  test  if  properly  performed.  One  of 
the  most  common  mistakes  in  the  manipulation  of  this  test  is 
the  use  of  a  guaiac  solution  which  is  too  concentrated  and 
which,  when  brought  into  contact  with  the  aqueous  blood  solu- 
tion, causes  the  separation  of  a  voluminous  precipitate  of  a 
resinous  material  which  may  obscure  the  blue  coloration;  this 
is  particularly  true  of  the  test  when  used  for  the  examination 
of  blood  stains.  A  solution  of  guaiac  made  by  dissolving  1 
gram  of  the  resin  in  60  c.c.  of  95  per  cent  alcohol  is  very 
satisfactory  for  general  use.  The  test  is  frequently  objected 
to  upon  the  ground  that  various  other  substances,  e.  g.,  milk, 
pus,  saliva,  etc.,  respond  to  the  test  and  that  it  cannot  therefore 


BLOOD.  159 

Ik.-  considered  a  specific  tc-t  for  bl 1  and  is  of  value  only  in  a 

negative  sense  We  have  demonstrated  to  our  own  satis- 
faction, however,  that  milk  many  times  gives  the  blue  color 
upon  the  addition  of  an  alcoholic  solution  of  guaiac  resin 
without  the  addition  of  hydrogen  peroxide  or  old  turpentine. 
Buckmaster  has  very  recently  advocated  the  use  of  an  alco- 
holic solution  of  guaiaconic  acid  instead  of  an  alcoholic  solu- 
tion of  guaiac  resin.  He  claims  that  he  was  able  to  produce 
the  blue  color  upon  the  addition  of  the  guaiaconic  acid  to 
milk  only  when  the  sample  of  milk  tested  was  brought  from 
the  country  in  sterile  bottles,  and  further,  that  no  sample  of 
London  milk  which  he  examined  responded  to  the  test.  In 
the  application  of  the  guaiac  test  to  the  detection  of  blood, 
he  states  that  he  was  able  to  detect  laked  blood  when  present 
in  the  ratio  1  :  5,000,000  and  unlaked  blood  when  present  in 
the  ratio  1 :  1,000,000.  This  author  considers  the  guaiac  test 
to  be  far  more  trustworthy  than  is  generally  believed. 

Up  to  within  very  recent  times  it  has  been  impossible  to 
make  an  absolute  differentiation  of  human  blood.  Recently 
however  the  so-called  "  biological  "  blood  test  has  made  such 
a  differentiation  possible.  This  test,  known  as  the  Bordet 
reaction,  is  founded  upon  the  fact  that  the  blood  serum  of  an 
animal  into  which  has  been  injected  the  blood  of  another  ani- 
mal of  different  species  develops  the  property  of  agglutinating 
and  dissolving  erythrocytes  similar  to  those  injected,  but  ex- 
erts this  influence  upon  blood  from  no  other  species.  The 
antiserum  used  in  this  test  is  prepared  by  injecting  rabbits 
with  5-10  c.c.  of  human  defibrinated  blood,  at  intervals  of 
about  four  days  until  a  total  of  between  50  and  80  c.c.  has  been 
injected.  After  a  lapse  of  one  or  two  weeks  the  animal  is 
bled,  the  serum  collected,  placed  in  sterile  tubes  and  preserved 
for  use  as  needed.  In  examining  any  specific  solution  for 
human  blood  it  is  simply  necessary  to  combine  the  antiserum 
and  the  solution  under  examination  in  the  proportion  of  1  :  100 
and  place  the  mixture  at  370  C.  If  human  blood  is  present 
in  the  solution  a  turbidity  will  be  noted  and  this  will  change 


l6o  PHYSIOLOGICAL    CHEMISTRY. 

within  three  hours  to  a  distinctly  flocculent  precipitate.     This 
antiserum  will  react  thus  with  no  other  known  substance. 

Experiments  on  Blood. 

I.    Defibrinated  Ox-blood. 

1.  Reaction. — Moisten  red  and  blue  litmus  papers  with  10 
per  cent  sodium  chloride  solution  and  test  the  reaction  of  the 
defibrinated  blood. 

2.  Microscopical  Examination. — Examine  a  drop  of  defi- 
brinated blood  under  the  microscope.  Compare  the  objects  you 
observe  with  Plate  IV,  opposite  page  151.  Repeat  the  test 
with  a  drop  of  your  own  blood. 

3.  Specific  Gravity. — Determine  the  specific  gravity  of 
defibrinated  blood  by  means  of  an  ordinary  specific  gravity 
spindle.  Compare  this  result  with  the  specific  gravity  as  de- 
termined by  Hammerschlag's  method  in  the  next  experiment. 

4.  Specific  Gravity  by  Hammerschlag's  Method. — Fill  an 
ordinary  urinometer  cylinder  about  one-half  full  of  a  mixture 
of  chloroform  and  benzene,  having  a  specific  gravity  of 
approximately  1.050.  Into  this  mixture  allow  a  drop  of  the 
blood  under  examination  to  fall  from  a  pipette  or  directly  from 
the  finger  in  case  fresh  blood  is  being  examined.  Care  must 
be  taken  not  to  use  too  large  a  drop  of  blood  and  to  keep  the 
drop  from  coming  in  contact  with  the  walls  of  the  cylinder. 
If  the  blood  drop  sinks  to  the  bottom  of  the  vessel,  thus  show- 
ing it  to  be  of  higher  specific  gravity  than  the  surrounding 
fluid,  add  chloroform  until  the  blood  drop  remains  suspended 
in  the  mixture.  Stir  carefully  with  a  glass  rod  after  adding 
the  chloroform.  If  the  blood  drop  rises  to  the  surface  upon 
being  introduced  into  the  mixture,  thus  showing  it  to  be  of 
lower  specific  gravity  than  the  surrounding  fluid,  add  benzene 
until  the  blood  drop  remains  suspended  in  the  mixture.  Stir 
with  a  glass  rod  after  the  benzene  is  added.  After  the  blood 
drop  has  been  brought  to  a  suspended  position  in  the  mixture 
by  means  of  one  or  more  additions  of  chloroform  and  benzene 
this  final  mixture  should  be  filtered  through  muslin  and  its 


BLOOD.  l6l 

specific  gravity  accurately  determined.     What  is  the  specific 
gravity  of  the  blood  under  examination? 

5.  Tests  for  Various  Constituents. — Place  10  c.c.  of  de- 
fibrinated  blood  in  an  evaporating  dish,  dilute  with  100  c.c.  of 
water  and  heat  to  boiling.  Is  there  any  coagulation,  and  if  so 
what  bodies  form  the  coagulum?  At  the  boiling-point  acidu- 
late slightly  with  dilute  acetic  acid.  Filter.  The  filtrate  should 
be  clear  and  the  coagulum  dark  brown.  Reserve  this  coag- 
ulum. What  body  gives  the  coagulum  this  color?  Evapo- 
rate the  filtrate  to  about  25  c.c,  filtering  off  any  precipitate 
which  may  form  in  the  process.  Make  the  following  tests 
upon  the  filtrate : 

(a)  Fe tiling's  Test. — Test  for  sugar  according  to  directions 
given  on  page  8. 

(b)  Chlorides. — To  a  small  amount  of  the  filtrate  in  a  test- 
tube  add  a  few  drops  of  nitric  acid  and  a  little  argentic  nitrate. 
In  the  presence  of  chlorides,  a  white  precipitate  of  argentic 
chloride  will  form. 

(c)  Phosphates. — Test  for  phosphates  by  nitric  acid  and 
molybdic  solution  according  to  directions  given  on  page  37. 

((/)  Proteose  and  Peptone. — Test  a  small  amount  of  the 
solution  for  proteose  and  peptone  by  saturating  with  ammo- 
nium sulphate  according  to  directions  given  on  page  59. 

(e)  Crystallization  of  Sodium  Chloride. — Place  the  re- 
mainder of  the  filtrate  in  a  watch  glass  and  evaporate  it  on  a 
water-bath.  Examine  the  crystals  under  the  microscope  and 
compare  them  with  those  in  Fig.  60,  page  167. 

6.  Test  for  Iron. — Incinerate  a  small  portion  of  the  coagu- 
lum from  the  last  experiment  ( 5 )  in  a  porcelain  crucible.  Cool, 
dissolve  the  residue  in  dilute  hydrochloric  acid  and  test  for  iron 
by  potassium  ferrocyanide  or  ammonium  sulphocyanide. 
Which  of  the  constituents  of  the  blood  contains  the  iron? 

7.  Laky  Blood. — Note  the  opacity  of  ordinary  defibrinated 
blood.  Place  a  few  cubic  centimeters  of  this  blood  in  a  test- 
tube  and  add  water,  a  little  at  a  time,  until  the  blood  is  ren- 
dered transparent.    It  is  now  laky  blood.    How  does  the  water 


1 62  PHYSIOLOGICAL    CHEMISTRY. 

act  in  causing  this  transparency?  Examine  a  drop  of  laky- 
blood  under  the  microscope.  How  does  its  microscopical  ap- 
pearance differ  from  that  of  unaltered  blood?  What  other 
agents  may  be  used  to  render  blood  laky? 

Fig.  57- 


\ 


Effect  of  Water  on  Erythrocytes. 

8.  Osmotic  Pressure. — Place  a  few  cubic  centimeters  of 
blood  in  each  of  three  test-tubes.  Lake  the  blood  in  the  first 
tube  according  to  directions  given  in  the  last  experiment  (7)  : 
add  an  equal  volume  of  isotonic  (0.9  per  cent)  sodium  chlo- 
ride to  the  blood  in  the  second  tube,  and  an  equal  volume  of 
10  per  cent  sodium  chloride  to  the  blood  in  the  third  tube. 
Mix  thoroughly  by  shaking  and  after  a  few  moments  examine 
a  drop  from  each  of  the  three  tubes  under  the  microscope 
(see  Figs.  57  and  115.  pages  162  and  337).  What  do  you 
find  and  what  is  your  explanation  from  the  standpoint  of 
osmotic  pressure? 

9.  Diffusion  of  Haemoglobin. — Prepare  some  laky  blood, 
thus  liberating  the  haemoglobin  from  the  erythrocytes.  Test 
the  diffusion  of  the  haemoglobin  by  preparing  a  dialyzer  like 
one  of  the  models  shown  in  Fig.  1,  page  6.  How  does  haemo- 
globin differ  from  other  well-known  crystallizable  bodies? 


BLOOD.  [63 

10.  Guaiac  Test. — To  5  c.c.  of  water  in  a  tesl  tube  add  two 
drops  of  blood.  By  means  of  a  pipette  drop  an  alcoholic 
solution  of  guaiac  (strength  about  [:6b)1  into  the  resulting 
mixture  until  a  turbidity  is  observed  and  add  old  turpentine  or 
hydrogen  pert  ixide,  dr<  >p  by  drop,  until  a  blue  c<  dor  is  obtained. 
Do  any  other  substances  respond  in  a  similar  manner  to  this 
test?  Is  a  positive  guaiac  test  a  sure  indication  of  the  p 
ence  of  blood? 

11.  Haemin  Test. —  (a)  Teichmanris  Method. —  Place  a 
very  small  drop  of  blood  on  a  microscopic  slide,  add  a  minute 
grain  of  sodium  chloride2  and  carefully  evaporate  to  dryness 
over  a  low  flame.  Put  a  cover  glass  in  place,  run  underneath 
it  a  drop  of  glacial  acetic  acid  and  warm  gently  until  the  for- 
mation of  gas  bubbles  is  noted.  Cool  the  preparation,  exam- 
ine under  the  microscope  and  compare  the  crystals  with  those 
shown  in  Figs.  58  and  59,  page  164.  The  haemin  crystals 
result  from  the  decomposition  of  the  haemoglobin  of  the  blood. 
What  are  the  steps  involved  in  this  process?  The  haemin 
crystals  are  also  called  Teichmann's  crystals.  Is  this  an 
absolute  test  for  blood?  Is  it  possible  to  differentiate  be- 
tween human  blood  and  the  blood  of  other  species  by  means 
of  the  haemin  test? 

(b)  Zeynek  and  Nencki's  Method. — To  10  c.c.  of  defibri- 
nated  blood  add  acetone  until  no  more  precipitate  forms. 
Filter  off  the  precipitated  proteid  and  extract  it  with  10  c.c.  of 
acetone  made  acid  with  2-3  drops  of  hydrochloric  acid.  Place 
a  drop  of  the  resulting  colored  extract  on  a  slide,  immediately 
place  a  cover  glass  in  position  and  examine  under  the  micro- 
scope. Upon  the  evaporation  of  the  acetone,  crystals  of  haemin 
will  form.  Larger  crystals  may  be  obtained  by  evaporating 
the  acetone  extract  about  one-half,  transferring  it  to  a  stop- 
pered vessel  and  allowing  it  to  remain  over  night. 

(c)  Schalfijew's  Method. — Place  20  c.c.   of  glacial  acetic 

1  Buckmaster   advises   the    use   of   an    alcoholic    solution    of   guaiaconic 
acid  instead  of  an  alcoholic  solution  of  guaiac  resin. 
J  Buckmaster  considers  the  use  of  potassium  chloride  preferable. 


164  PHYSIOLOGICAL    CHEMISTRY. 

Pic.   58. 

Hjemin   Crystals  from  Human   Blood. 

Reproduced  from   a   micro-photograph   furnished  by  Prof.   E.  T.   Reichert,   of 

the  University   of   Pennsylvania. 


Fig.  59. 


H;emin  Crystals  from  Sheep  Blood. 

Reproduced  from   a  micro-photograph   furnished  by  Prof.   E.  T.  Reichert,  of 

the  University   of   Pennsylvania. 


BLOOD.  165 

acid  in  a  small  beaker  and  heat  to  8oc  C.  Add  5  c.c.  of 
strained  defibrinated  blood,  again  bring  the  temperature  to 
80  C,  remove  the  flame  and  allow  the  mixture*  to  cool. 
Examine  the  crystals  under  the  microscope  and  compare  them 
with  those  reproduced  in  Figs.  58  and  59,  page  1^4. 

12.  Catalytic  Action. — To  about  10  drops  of  blood  in  a 
test-tube  add  twice  the  volume  of  hydrogen  peroxide,  without 
shaking.  The  mixture  foams.  What  is  the  cause  of  this 
phenomenon  ? 

[3.  Preparation  of  Haematin. — Place  100  c.c.  of  laked 
bli  k  m1  in  a  beaker  and  add  95  per  cent  alcohol  until  precipitation 
ceases.  What  bodies  are  precipitated?  Transfer  the  precipi- 
tate to  a  flask  and  boil  with  95  per  cent  alcohol  previously 
acidulated  with  sulphuric  acid.  Through  the  action  of  the  acid 
the  haemoglobin  is  split  into  haematin  and  a  proteid  body  called 
globin*.  Later  the  "sulphuric  acid  ester  of  haematin"  is 
formed,  which  is  soluble  in  the  alcohol.  Continue  heating 
until  the  precipitate  is  no  longer  colored,  then  filter.  Partly 
saturate  the  filtrate  with  sodium  chloride  and  warm.  In  this 
process  the  "  hydrochloric  acid  ester  of  haematin  "  is  formed. 
Filter  and  dissolve  on  the  filter  paper  by  sodium  carbonate. 
Save  this  alkaline  solution  of  haematin  and  make  a  spectro- 
scopic examination  later  after  becoming  familiar  with  the  use 
of  the  spectroscope.  How  does  the  spectrum  of  oxyhemo- 
globin differ  from  that  of  the  derived  alkali  hcematin? 

14.  Variation  in  Size  of  Erythrocytes. — Prepare  two 
small  funnels  with  filter  papers  such  as  are  used  in  quantitative 
analysis.  Moisten  each  paper  with  normal  (isotonic)  salt 
solution.  Into  one  funnel  introduce  a  small  amount  of  defibri- 
nated ox  blood  and  into  the  other  funnel  allow  blood  to  drop 
directly  from  a  decapitated  frog.  Note  that  the  filtrate  from 
the  ox  blood  is  colored  whereas  that  from  the  frog  blood  is 
colorless.  What  deduction  do  you  make  regarding  the  relative 
size  of  the  erythrocytes  in  ox  and  frog  blood?  Does  either 
filtrate  clot?     Why? 


1 66  PHYSIOLOGICAL    CHEMISTRY. 

II.    Blood  Serum. 

1.  Coagulation  Temperature. — Place  5  c.c.  of  undiluted 
serum  in  a  test-tube  and  determine  its  temperature  of  coagula- 
tion according  to  the  method  described  on  page  50.  Note  the 
temperature  at  which  a  cloudiness  occurs  as  well  as  the  tem- 
perature at  which  coagulation  is  complete. 

2.  Precipitation  by  Alcohol. — To  5  c.c.  of  serum  in  a 
test-tube  add  twice  the  amount  of  95  per  cent  alcohol  and 
thoroughly  mix  by  shaking.  What  is  this  precipitate?  Make 
a  confirmatory  test.  Test  the  alcoholic  filtrate  for  proteid. 
Explain  the  result. 

3.  Proteids  of  Blood  Serum. — Place  about  20  c.c.  of  un- 
diluted serum  in  a  small  evaporating  dish,  heat  to  boiling  and 
at  the  boiling-point  acidify  slightly  with  dilute  acetic  acid.  Of 
what  does  this  coagulum  consist?  Filter  off  the  coagulum 
(reserve  the  filtrate)  and  test  it  as  follows: 

(a)  Millons  Reaction. — Make  the  test  according  to  direc- 
tions given  on  page  44. 

(b)  Xanthoproteic  Test. — Make  the  test  according  to  di- 
rections given  on  page  44. 

4.  Sugar  in  Serum. — Test  a  little  of  the  filtrate  from  Ex- 
periment 3  by  Fehling's  test.     What  do  you  conclude  ? 

5.  Detection  of  Sodium  Chloride. —  (a)  Test  a  little  of 
the  filtrate  from  Experiment  3  for  chlorides,  by  the  use  of 
nitric  acid  and  argentic  nitrate,  (b)  Evaporate  5  c.c.  of  the 
filtrate  from  Experiment  3  in  a  watch  glass  on  a  water-bath. 
Examine  the  crystals  and  compare  them  with  those  reproduced 
in  Fig.  60,  page  167. 

6.  Separation  of  Serum  Globulin  and  Serum  Albunin. 
— Place  10  c.c.  of  blood  serum  in  a  small  beaker  and  saturate 
with  magnesium  sulphate.  What  is  this  precipitate?  Filter 
it  off  and  acidify  the  filtrate  slightly  with  acetic  acid.  What 
is  this  second  precipitate?  Filter  this  precipitate  off  and  test 
the  filtrate  by  the  biuret  test.     What  do  you  conclude  ? 


167 


£#30 


Sodium  Chloride. 
III.    Blood  Plasma. 

i.  Preparation  of  Oxalated  Plasma. — Allow  arterial 
blood  to  run  into  an  equal  volume  of  0.2  per  cent  ammonium 
oxalate  solution. 

2.  Preparation  of  Fibrinogen. — To  25  c.c.  of  oxalated 
plasma  add  an  equal  volume  of  saturated  sodium  chloride  solu- 
tion. Note  the  precipitation  of  fibrinogen.  Filter  off  the  pre- 
cipitate (reserve  the  filtrate)  and  test  it  by  a  proteid  color  test 
I  see  page  44). 

3.  Effect  of  Calcium. — Place  a  small  amount  of  oxalated 
plasma  in  a  test-tube  and  add  a  few  drops  of  a  2  per  cent 
calcium  chloride  solution.     What  occurs?    Explain  it. 

4.  Preparation  of  Salted  Plasma. — Allow  arterial  blood 
to  run  into  an  equal  volume  of  a  saturated  solution  of  sodium 
sulphate  or  a  10  per  cent  solution  of  sodium  chloride.  Keep 
the  mixture  in  a  cold  place  for  about  twenty-four  hours. 

5.  Effect  of  Dilution. — Place  a  few  drops  of  salted  plasma 
in  a  test-tube  and  dilute  it  with  10-15  volumes  of  water. 
What  do  you  observe?   Explain  it. 

6.  Crystallization  of  Oxyhemoglobin. — RcicJicrt's  Meth- 
od,— Allow  the  blood  of  the  dog  or  horse  to  Mow  into  an  equal 


1 68  PHYSIOLOGICAL    CHEMISTRY. 

volume  of  7  per  cent  ammonium  oxalate  solution.  Place  a 
small  amount  of  this  oxalated  blood  in  a  test-tube  and  lake  it 
with  ether,  being  careful  to  avoid  an  excess  of  the  reagent. 
By  means  of  a  pipette  transfer  a  drop  of  this  laked  blood  to  a 
slide,  and  when  the  edges  of  the  drop  begin  to  dry  place  a 
cover  glass  in  position.  Examine  under  the  microscope  and 
compare  the  crystals  with  those  in  Figs.  50  to  56,  pages  152 
t0  J55- 

IV.    Fibrin. 

1.  Preparation  of  Fibrin. — Allow  blood  to  flow  directly 
from  the  animal  into  a  vessel  and  rapidly  whip  it  by  means  of 
a  bundle  of  twigs,  a  mass  of  strong  cords  or  a  specially  con- 
structed beater.  If  a  pure  fibrin  is  desired  it  is  not  best  to 
attempt  to  manipulate  a  large  volume  of  blood  at  one  time. 
After  the  fibrin  has  been  collected  it  should  be  freed  from 
any  adhering  blood  clots  and  washed  in  water  to  remove  fur- 
ther traces  of  blood.  The  pure  product  should  be  very  light 
in  color.  It  may  be  preserved  under  glycerin,  dilute  alcohol 
or  chloroform  water. 

2.  Solubility. — Try  the  solubility  of  small  shreds  of  freshly 
prepared  fibrin  in  the  usual  solvents. 

3.  Millon's  Reaction. — Make  the  test  according  to  direc- 
tions given  on  page  44. 

4.  Xanthoproteic  Test. — Make  the  test  according  to  di- 
rections given  on  page  44. 

5.  Biuret  Test. — Make  the  test  according  to  directions 
given  on  page  45. 

V.    Detection  of  Blood  in  Stains  on  Cloth,  etc. 

i.  Identification  of  Corpuscles. — If  the  stain  under  ex- 
amination is  on  cloth  a  portion  should  be  extracted  with  a  few 
drops  of  glycerin  or  normal  (0.9  per  cent)  sodium  chloride 
solution.  A  drop  of  this  solution  should  then  be  examined 
under  the  microscope  to  determine  if  corpuscles  are  present. 

2.  Tests  on  Aqueous  Extract. — A  second  portion  of  the 
stain  should  be  extracted  with  a  small  amount  of  water  and 
the  following  tests  made  upon  the  aqueous  extract: 


BLOOD.  169 

(a)   HtBtnochrotnogen. — Make  a  small  amount  of  the  ex 
tract  alkaline  by  potassium  hydroxide  or  sodium  hydroxide, 

and  heat  until  a  brownish-green  color  results.  Cool  and  add 
a  few  drops  of  ammonium  sulphide  or  Stokes'  reagent  (see 
page  170)  and  make  a  spectroscopic  examination.  Compare 
the  spectrum  with  that  of  haemochromogen  (see  Absorption 
Spectra,  Plate  II). 

(/;)  Ilccmiii  Test. — Make  this  test  upon  a  small  drop  of 
the  aqueous  extract  according  to  the  directions  given  on  page 
163. 

(c)  Guaiac  Test. — Make  this  test  on  the  aqueous  extract 
according  to  the  directions  given  on  page  163.  The  guaiac 
solution  may  also  be  applied  directly  to  the  stain  without  pre- 
vious extraction  in  the  following  manner:  Moisten  the  stain 
with  water,  and  after  allowing  it  to  stand  several  minutes,  add 
an  alcoholic  solution  of  guaiac  (strength  about  1:60)  and  a 
little  hydrogen  peroxide  or  old  turpentine.  The  customary 
blue  color  will  be  observed  in  the  presence  of  blood. 

(d)  Acid  Hcematin. — If  the  stain  fails  to  dissolve  in  water 
extract  with  acid  alcohol  and  examine  the  spectrum  for  ab- 
sorption bands  of  acid  haematin  1  sec  Absorption  Spectra, 
Plate  II). 

VI.    Spectroscopic    Examination    of    Blood. 

(For  Absorption  Spectra  see  Plates  I.  and  II.). 

Either  the  awgtt/ar-vision  spectroscope  (Figs.  62  and  63, 
pages  170  and  171)  or  the  direct-vision  spectroscope  (Fig.  61, 
page  170)  may  be  used  in  making  the  spectroscopic  examina- 
tion of  the  blood.  For  a  complete  description  of  these  instru- 
ments the  student  is  referred  to  any  standard  text-book  of 
physics. 

1.  Oxyhaemoglobin. — Examine  dilute  (1:50)  defibrinated 
blood  spectroscopically.  Note  the  broad  absorption-band  be- 
tween D  and  E.  Continue  the  dilution  until  this  single  broad 
band  gives  place  to  two  narrow  bands,  the  one  nearer  the  D 
line  being  the  narrower.  These  are  the  typical  absorption- 
bands  of  oxyhemoglobin  obtained   from  dilute  solutions  of 


170 


PHYSIOLOGICAL    CHEMISTRY. 


blood.  Now  dilute  the  blood  very  freely  and  note  that  the 
bands  gradually  become  more  narrow  and,  if  the  dilution  is 
sufficiently  great,  they  finally  entirely  disappear. 


Fig.  61. 


Direct-vision   Spectroscope. 


2.  Haemoglobin  (so-called  Reduced  Haemoglobin). — To 
blood  which  has  been  diluted  sufficiently  to  show  well  defined 
oxyhemoglobin    absorption-bands    add    a    small    amount     of 


Fig.  62. 


Angular-vision   Spectroscope  Arranged  for  Absorption  Analysis. 


Stokes'  reagent.1  The  blood  immediately  changes  in  color 
from  a  bright  red  to  a  violet-red.  The  oxyhemoglobin  has 
been   reduced   through    the   action    of    Stokes'    reagent   and 

1  Stokes'  reagent  is  a  solution  containing  2  per  cent  ferrous  sulphate  and 
3  per  cent  tartaric  acid.  When  needed  for  use  a  small  amount  should  be 
placed  in  a  test-tube  and  ammonium  hydroxide  added  until  the  precipitate 
which  forms  on  the  first  addition  of  the  hydroxide  has  entirely  dissolved. 
This  produces  ammonium  ferrotartrate  which  is  a  reducing  agent. 


BLOOD. 


I7I 


haemoglobin  (so  called  reduced  haemoglobin)  has  been  formed. 
This  has  been  brought  about  by  the  removal  of  some  of  the 
loosely  combined  oxygen  from  the  oxyhemoglobin.  Examine 
this  haemoglobin  spectroscopically.  Note  that  in  place  of  the 
two  absorption  hands  of  oxyhemoglobin  we  now  have  a  single 
broad  hand  lying  almost  entirel)   between  1)  and  E.     This  is 

Fig.  63. 


Diagram  of  Angular-vision   Spectroscope.      (Long.) 

The  white  light  F  enters  the  collimator  tube  through  a  narrow  slit  and 
passes  to  the  prism  P,  which  has  the  power  of  refracting  and  dispersing  the 
light.  The  rays  then  pass  to  the  double  convex  lens  of  the  ocular  tube  and 
are  deflected  to  the  eyepiece  E.  The  dotted  lines  show  the  magnified  virtual 
image  which  is  formed.  The  third  tube  contains  a  scale  whose  image  is 
reflected  into  the  ocular  and  shown  with  the  spectrum.  Between  the  light 
F,  and  the  collimator  slit  is  placed  a  cell  to  hold  the  solution  undergoing 
examination. 


the  typical  spectrum  of  hemoglobin.  If  the  solution  showing 
this  spectrum  be  shaken  in  the  air  for  a  few  moments  it  will 
again  assume  the  bright  red  color  of  oxyhemoglobin  and  show 
the  characteristic  spectrum  of  that  pigment. 

3.  Carbon  Monoxide  Haemoglobin. — The  preparation  of 
this  pigment  may  be  easily  accomplished  by  passing  ordinary 
illuminating  gas1  through  defibrinated  ox-blood.  Blood  thus 
treated  assumes  a  brighter  tint  (carmine)  than  that  imparted 
by  oxyhemoglobin.     In  very  dilute  solution  oxyhemoglobin 

1  The  so-called  water  gas  with  which  ordinary  illuminating  gas  is  diluted 
contains  usually  as  much  as  20  per  cent  of  carbon  monoxide   (CO). 


172  PHYSIOLOGICAL    CHEMISTRY. 

appears  yellowish-red  whereas  carbon  monoxide  haemoglobin 
under  the  same  conditions  appears  bluish-red.  Examine- the 
carbon  monoxide  haemoglobin  solution  spectroscopically.  Ob- 
serve that  the  spectrum  of  this  body  resembles  the  spectrum  of 
oxyhemoglobin  in  showing  two  absorption-bands  between  D 
and  E.  The  bands  of  carbon  monoxide  haemoglobin,  however, 
are  somewhat  nearer  the  violet  end  of  the  spectrum.  Add 
some  Stokes'  reagent  to  the  solution  and  again  examine  spec- 
troscopically. Note  that  the  position  and  intensity  of  the 
absorption  bands  remain  unaltered. 

4.  Neutral  Methaemoglobin. — Dilute  a  little  defibrinated 
blood  (1  :io)  and  add  a  few  drops  of  a  freshly  prepared  10 
per  cent  solution  of  potassium  ferricyanide.  Shake  this  mix- 
ture and  observe  that  the  bright  red  color  of  the  blood  is  dis- 
placed by  a  brownish-red.  Now  dilute  a  little  of  this  solution 
and  examine  it  spectroscopically.  Note  the  single,  very  dark 
absorption-band  lying  to  the  left  of  D  and,  if  the  dilution  is 
sufficiently  great,  also  observe  the  two  rather  faint  bands  lying 
between  D  and  E  in  somewhat  similar  positions  to  those  occu- 
pied by  the  absorption-bands  of  oxyhaemoglobin.  Add  a  few 
drops  of  Stokes'  reagent  to  the  methaemoglobin  solution  while 
it  is  in  position  before  the  spectroscope  and  note  the  imme- 
diate appearance  of  the  oxyhaemoglobin  spectrum  which  is 
quickly  followed  by  that  of  haemoglobin. 

5.  Alkaline  Methaemoglobin. — Render  a  neutral  solution 
of  methaemoglobin,  such  as  that  used  in  the  last  experiment 
(4),  slightly  alkaline  with  a  few  drops  of  ammonia.  The  solu- 
tion becomes  redder  in  color,  due  to  the  formation  of  alkaline 
methaemoglobin  and  shows  a  spectrum  different  from  that  of 
the  neutral  body.  In  this  case  we  have  a  band  on  either  side 
of  D,  the  one  nearer  the  red  end  of  the  spectrum  being  much 
the  fainter.  A  third  band,  darker  than  either  of  those  men- 
tioned lies  between  D  and  E  somewhat  nearer  E. 

6.  Alkali  Haematin. — Observe  the  spectrum  of  the  alkali 
haematin  prepared  in  Experiment  13  on  page  165.  Also  make 
a    spectroscopic    examination    of    a    freshly    prepared    alkali 


BLOOD.  [73 

haematin.1  The  typical  spectrum  of  alkali  haematin  shows  a 
single  absorption  band  lying  across  D  and  mainly  toward  the 
red  end  of  the  spectrum. 

7.  Reduced  Alkali  Haematin  or  Haemochromogen. — 
Dilute  the  alkali  1 1  a  *  n  1  a  t  i  1 1  solution  used  in  the  last  experiment 
(6)  to  such  an  extent  that  it  shows  no  absorption  hand.  Now 
add  a  few  drops  of  Stokes'  redgent  and  note  that  the  greenish- 
brown  color  of  the  alkali  haematin  solution  is  displaced  by  a 
bright  red  color.  This  is  due  to  the  formation  of  haemochro- 
mogen or  reduced  alkali  haematin.  Examine  this  solution 
spectroscopically  and  observe  the  narrow,  dark,  absorption- 
band  lying  midway  between  D  and  E.  If  the  dilution  is  not 
too  great  a  faint  band  may  be  observed  in  the  green  extend- 
ing across  E  and  b. 

8.  Acid  Haematin. — To  some  defibrinated  blood  add  half 
its  volume  of  glacial  acetic  acid  and  an  equal  volume  of  ether. 
Mix  thoroughly.  The  acidified  ethereal  solution  of  haematin 
rises  to  the  top  and  may  be  poured  off  and  used  for  the  spec- 
troscopic examination.  If  desired  it  may  be  diluted  with 
acidified  ether  in  the  ratio  of  one  part  of  glacial  acetic  acid 
to  two  parts  of  ether.  A  distinct  absorption-band  will  be 
noted  in  the  red  between  C  and  D  and  lying  somewhat  nearer 
C  than  the  band  in  the  metbsemoglobin  spectrum.  Between  D 
and  F  may  be  seen  a  rather  indistinct  broad  band.  Dilute  the 
solution  until  this  band  resolves  itself  into  two  bands.  Of 
these  the  more  prominent  is  a  broad,  dark  absorption-band 
lying  in  the  green  between  b  and  F.  The  second,  a  narrow 
band  of  faint  outline,  lies  in  the  light  green  to  the  red  side 
of  E.  A  fourth  very  faint  band  may  be  observed  lying  on  the 
violet  side  of  D. 

9.  Acid  Haematoporphyrin. — To  5  c.c.  of  concentrated 
sulphuric  acid  in  a  test-tube  add  two  drops  of  blood  mixing 

1  Alkali  haematin  may  be  prepared  by  mixing  one  volume  of  a  con- 
centrated potassium  hydroxide  or  sodium  hydroxide  solution  and  two  vol- 
umes of  dilute  (1:5)  defibrinated  blood.  This  mixture  should  be  heated 
gradually  almost  to  boiling,  then  cooled  and  shaken  for  a  few  moments 
in  the  air  before  examination. 


174  PHYSIOLOGICAL    CHEMISTRY. 

thoroughly  by  agitation  after  the  addition  of  each  drop.  A 
wine-red  solution  is  produced.  Examine  this  solution  spectro- 
scopically.  Acid  haematoporphyrin  gives  a  spectrum  with  an 
absorption-band  on  either  side  of  D,  the  one  nearer  the  red 
end  of  the  spectrum  being  the  narrower. 

10.  Alkaline  Haematoporphyrin. — Introduce  the  acid 
haematoporphyrin  solution  just  examined  into  an  excess  of 
distilled  water.  Cool  the  solution  and  add  potassium  hydrox- 
ide slowly  until  the  reaction  is  but  slightly  acid.  A  colored 
precipitate  forms  which  includes  the  principal  portion  of  the 
haematoporphyrin.  The  presence  of  sodium  acetate  facili- 
tates the  formation  of  this  precipitate.  Filter  off  the  precipi- 
tate and  dissolve  it  in  a  small  amount  of  dilute  potassium 
hydroxide.  Alkaline  haematoporphyrin  prepared  in  this  way 
forms  a  bright  red  solution  and  possesses  four  absorption- 
bands.  The  first  is  a  very  faint,  narrow  band  in  the  red, 
midway  between  C  and  D;  the  second  is  a  broader,  darker 
band  lying  across  D,  principally  to  the  violet  side.  The  third 
absorption-band  lies  principally  between  D  and  E,  extending 
for  a  short  distance  across  E  to  the  violet  side,  and  the  fourth 
band  is  broad  and  dark  and  lies  between  b  and  F.  The  first 
band  mentioned  is  the  faintest  of  the  four  and  is  the  first  to 
disappear  when  the  solution  is  diluted. 

VII.    Instruments  Used  in  the  Clinical  Examination  of  the  Blood. 

i.  Fleischl's  Haemometer  (Fig.  64,  p.  175). — This  is  an 
instrument  used  quite  extensively  clinically,  for  the  quantitative 
determination  of  haemoglobin.  The  instrument  consists  of  a 
small  cylinder  which  is  provided  with  a  fixed  glass  bottom  and 
a  movable  glass  cover,  and  which  is  divided,  by  means  of  a 
metal  septum,  into  two  compartments  of  equal  capacity.  This 
cylinder  is  supported  in  a  vertical  position  by  means  of  a  mech- 
anism which  resembles  the  base  and  stage  of  an  ordinary  mi- 
croscope. Underneath  the  stage  is  placed  a  colored  glass  wedge 
(see  Fig.  66,  p.  176),  so  arranged  as  to  run  immediately 
beneath  the  glass  bottom  of  one  of  the  compartments  of  the 


BLOOD. 


175 


Fig,  ''I 


cylinder  and  ground  in  such  a  manner  that  each  part  of  the 
wedge  corresponds  in  color  to  a  solution  of  haemoglobin  of 
some  definite  percentage.  The  glass  wedge  is  held  in  a  metal 
frame  and  may  be  moved  backward  or  forward  by  means  of  a 
rack  and  pinion  arrangement.  A  scale  along  the  side  ol  this 
frame  indicates  the  percentage  of 
the  normal  amounl  of  haemoglo- 
bin which  each  particular  varia- 
tion in  the  depth  of  color  of  the 
ground  wedge  represents,  tak- 
ing the  normal  haemoglobin  con- 
tent as  ioo.1  In  a  position 
corresponding  to  the  position  of 
the  mirror  on  the  ordinary  mi- 
croscope is  attached  a  light- 
colored  opaque  plate  which 
serves  to  reflect  the  light  up- 
ward through  the  colored  wedge 
and  the  cylinder  to  the  eye  of 
the  observer. 

In  making  a  determination  of  the  percentage  of  haemo- 
globin by  this  instrument  the  procedure  is  as  follows :  Fill  each 
compartment  about  three-fourths  full  of  distilled  water.  Punc- 
ture the  finger-tip  or  lobe  of  the  ear 
of  the  subject  by  means  of  a  sterile 
needle  or  scalpel  and,  as  soon  as  a 
drop  of  blood  appears,  place  one  end 
of  the  capillary  pipette  (Fig.  65), 
which  accompanies  the  instrument, 
against  the  drop  and  allow  it  to  fill  by 
capillary  attraction.  To  prevent  the 
blood  from  adhering  to  the  exterior 
of  the  tube,  and  so  render  the  deter- 
mination inaccurate,  it  is  customary  to  apply  a  very  thin 
coating  of  mutton  fat  to  the  outer  surface  before  using  or  to 
1  The  scale  of  the  ordinary  instrument  is  usually  too  high. 


Fl.l   !  S<    III.'S       H.VMOMF/I  ER. 

(Da    Costa.) 


Fig.  65. 


Pipette  of  Fleischl's 

HiEMOMETER. 


176 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  66. 


Colored    Glass    Wedge    of   Fleischl's 
H^emometer.      (Da    Costa.) 


wrap  the  tube  in  a  piece  of  oily  chamois  when  not  in  use.  As 
soon  as  the  tube  has  been  accurately  filled  with  blood  it  should 
be  dipped  into  the  water  of  one  of  the  compartments  of  the 
cylinder  and  all  traces  of  the  blood  washed  out  with  water  by 
means  of  a  small  dropper  which  accompanies  the  instrument. 

If  the  blood  is  not  well  dis- 
tributed throughout  the 
compartment  and  does  not 
form  a  homogeneous  solu- 
tion the  contents  of  the  com- 
partment should  be  mixed 
thoroughly  by  means  of  the 
metal  handle  of  the  cap- 
illary measuring  pipette. 
When  this  has  been  done  each  compartment  should  be  completely 
filled  with  distilled  water  and  the  glass  cover  adjusted,  care 
being  taken  that  the  contents  of  the  two  compartments  do  not 
mix.  Now  adjust  the  cylinder  so  that  the  compartment  con- 
taining the  pure  distilled  water  is  immediately  above  the  colored 
glass  wedge.  By  means  of  the  rack  and  pinion  arrangement, 
manipulate  the  colored  wedge,  until  a  portion  of  it  is  found 
which  corresponds  in  color  with  the  diluted  blood.  When  this 
agreement  in  color  has  been  secured  the  point  on  the  scale 
corresponding  to  this  particular  color  should  be  read  and  the 
actual  percentage  of  haemoglobin  computed.  For  instance,  if 
the  scale  reading  is  90  it  means  that  the  blood  under  examina- 
tion contains  90  per  cent  of  the  normal  quantity  of  haemo- 
globin, i.  e.,  90  per  cent  of  14  per  cent. 

2.  Fleischl-Miescher  Haemometer. — The  apparatus  of 
Fleischl  has  recently  been  modified  by  Miescher.  If  all  pre- 
cautions are  taken,  the  margin  of  error  in  the  absolute  quan- 
tity of  haemoglobin  determined  by  this  instrument  does  not 
exceed  0.15-0.22  per  cent  by  weight  of  the  blood.  Detailed 
directions  for  the  manipulation  of  the  Fleischl-Miescher 
haemometer  accompany  the  instrument.  In  brief  Miescher 
modified  the  instrument  as  follows :   ( 1 )   The  scale  of  each 


BLOOD.  177 

instrument  is  supplied  with  a  caliber  table  of  absolute  haemo- 
globin values,  expressed  in  milligrams:  the  ^cale  of  Fleischl's 
haemometer  shows  the  percentage  of  haemoglobin  in  relation 
to  an  average  selected  somewhat  arbitrarily.  Thus  many 
errors  arising  from  the  irregular  coloring  of  the  glass  wedge 
of  the  older  apparatus  are  avoided  in  the  instrument  as  modi- 
fied. (2)  Bach  instrument  is  accompanied  by  a  measuring 
pipette  (melangeur)  which  allows  of  a  more  accurate  meas- 
urement of  the  blood  than  was  possible  with  the  capillary  tubes 
of  the  older  apparatus.  (3)  With  the  aid  of  the  measuring 
pipette  mentioned  above  blood  of  varying  degrees  of  con- 
centration may  be  compared.  In  this  way  the  individual  ex- 
aminations are  controlled  and  a  check  upon  the  accuracy  of  the 
graduation  in  the  color  of  the  glass  wedge  is  also  afforded. 
This  wedge  is  much  more  evenly  and  accurately  colored  than 
in  the  unmodified  apparatus  of  Fleischl.  (4)  Before  reading 
the  percentage  as  indicated  by  the  scale,  the  chamber  is  covered 
with  a  glass  and  a  diaphragm  which  sharply  define  the  field 
on  all  sides  without  the  formation  of  a  meniscus. 

The  measuring  pipette  is  constructed  essentially  the  same 
as  the  pipettes  which  accompany  the  Thoma-Zeiss  Apparatus 
1  see  p.  [82).  The  capillary  portion,  however,  is  graduated  I, 
Yz  and  l/>  which  enables  the  observer  to  dilute  the  blood 
sample  in  the  proportion  of  1  :200,  1  1300  or  1  1400  as  he  may 
desire.  If  there  is  difficulty  in  drawing  in  the  blood  exactly 
to  one  of  the  graduations  just  mentioned  the  amount  of  blood 
above  or  below  the  volume  indicated  by  the  graduation  may 
be  determined  by  means  of  certain  delicate  cross-lines  which 
are  placed  directly  above  and  below  the  graduation.  Each 
cross-line  corresponds  to  T^  of  the  volume  of  the  capillary 
tube  from  the  tip  to  the  1  graduation. 

A  0.1  per  cent  solution  of  sodium  carbonate  is  used  to  dis- 
solve the  stroma  of  the  erythrocytes  and  so  render  the  blood 
solution  perfectly  clear.  If  this  is  not  done  the  color  of  the 
blood  solution  invariably  appears  darker  in  tone  than  that  of 
the  colored  glass  wedge.  A  freshly  prepared  sodium  carbo- 
•3 


178 


PHYSIOLOGICAL    CHEMISTRY 


nate  solution  should  be  used  in  order  that  the  clearness  of 
the  solution  may  not  be  marred  by  the  presence  of  sodium 
bicarbonate. 

3.  Dare's  Haemoglobinometer  (Fig.  67,  below). — This 
instrument,  as  the  name  signifies,  is  used  for  the  determina- 
tion of  haemoglobin.  In 
using  either  Fleischl's 
hremometer  or  the  in- 
strument as  modified  by 
Miescher  the  blood  is  di- 
luted for  examination 
whereas  with  the  Dare 
instrument  no  dilution  is 
required.  This  probably 
allows  of  rather  more 
accurate  determinations 
than  are  possible  with 
the  old  Fleischl  appa- 
ratus. 

The  instrument  con- 
sists essentially  of  the  fol- 
lowing parts  :  ( 1 )  A  cap- 
illary observation  cell, 
(2)  a  semicircular  col- 
ored glass  wedge,  (3)  a 
milled  wheel  for  manip- 
ulating the  wedge,  (4) 
a  candle  used  to  illumi- 
nate portions  of  the  cap- 
illary observation  cell 
and  the  colored  wedge, 
(5)  a  small  telescope  used  in  the  examination  of  the  areas 
illuminated  by  the  candle  flame,  (6)  a  scale  graduated  in  per- 
centages of  the  normal  amount  of  haemoglobin,  (7)  a  hard 
rubber  case,  (8)  a  movable  screen  attached  to  the  case. 

The   capillary   observation   cell    is    formed   of   two    small, 


Dare's    Haemoglobinometer.       (Da    Costa.) 

R,  Milled  wheel  acting  by  a  friction 
bearing  on  the  rim  of  the  color  disc ; 
S,  case  inclosing  color  disc,  and  provided 
with  a  stage  to  which  the  blood  cham- 
ber is  fitted ;  T,  movable  wing  which 
is  swung  outward  during  the  observation, 
to  serve  as  a  screen  for  the  observer's  eyes, 
and  which  acts  as  a  cover  to  inclose  the 
color  disc  when  the  instrument  is  not  in 
use ;  U,  telescoping  camera  tube,  in  posi- 
tion for  examination  ;  V,  aperture  admitting 
light  for  illumination  of  the  color  disc ;  X, 
capillary  blood  chamber  adjusted  to  stage 
of  instrument,  the  slip  of  opaque  glass,  W, 
being  nearest  to  the  source  of  light ;  Y, 
detachable  candle-holder ;  Z,  rectangular  slot 
through  which  the  haemoglobin  scale  indi- 
cated on  the  rim  of  the  color  disc  is  read. 


lil.OOD. 


T79 


Fig.  68. 


polished  rectangular  plates  of  glass,  one  being  transparent 
and  the  other  opaque.  When  held  in  position  on  the  instru- 
ment, by  means  of  a  small  metal  bracket,  the  opaque  portion 
of  the  cell  is  nearer  the  candle  and  thus  serves  to  soften  the 
glare  of  light  when  an  observation  is  being  made.  The  trans- 
parent portion  of  the  cell  is  directly  over  a  circular  opening 
in  the  case,- through  which  the  blood  specimen  is  viewed  by 
means  of  the  small  telescope. 

The  semicircular  colored  glass  wedge  is  so  ground  that 
each  particular  shade  of  color  corresponds  to  that  possessed 
by  fresh  blood  which  contains  some  definite  percentage  of 
haemoglobin.  It  is  mounted  upon 
a  disc  which  may  he  manipulated 
by  the  milled  wheel  in  such  a 
manner  as  to  bring  successive 
portions  of  the  wedge  in  position 
to  be  viewed  through  a  circular 
opening  contiguous  to  the  opening 
through  which  the  blood  specimen 
is  viewed.  For  a  further  descrip- 
tion of  the  instrument  see  Figures 
67,  68  and  69,  on  pages  178,  179, 
and  180  respectively. 

In  using  the  Dare  hsemoglobi- 
nometer  proceed  as  follows:  Punc- 
ture the  finger-tip  or  lobe  of  the  ear 
of  the  subject  by  means  of  a  needle 
or  scalpel  and.  after  a  drop  of  blood 

of  good  proportions  has  formed,  place  the  flat  capillary  observa- 
tion cell  in  contact  with  the  drop  and  allow  it  to  fill  by  capillary 
attraction  (  Fig.  69,  page  180).  Replace  the  cell  in  its  proper 
place  on  the  instrument.  When  in  position,  a  portion  of  this 
cell  may  be  observed  through  a  small  telescope  attached  to  the 
apparatus.  It  is  viewed  through  a  circular  opening  and  near 
this  circle  is  a  second  one  through  which  a  portion  of  a  semi- 
circular colored  glass  wedge  is  visible.     These  two  circles  are 


Horizontal  Section  of  D 
FLemoglobinometer. 

(Da     Costa. ) 


l8o  PHYSIOLOGICAL    CHEMISTRY. 

illuminated  simultaneously  by  means  of  the  flame  of  a  candle. 
The  colored  glass  may  be  rotated  by  means  of  a  milled  wheel 
and  the  point  of  agreement  of  the  color  of  the  adjoining  discs 
may  be  determined  in  the  same  way  as  in  Fleischl's  haemom- 
eter.     The  scale  reading  gives  the  percentage  of  the  normal 

Fig.  69. 


Method  of  Filling  the  Capillary  Observation   Cell  of   Dare's 

H.EMOGLOBINOMETER.        (Da    Costa.) 

quantity  of  haemoglobin  which  the  blood  sample  under  exami- 
nation contains.  Compute  the  actual  haemoglobin  content  in 
the  same  manner  as  from  the  scale  reading  of  the  Fleischl 
haemometer  (seepage  176). 

4.  Tallquist's  Haemoglobin  Scale. — This  consists  essen- 
tially of  a  series  of  ten  colors  corresponding  to  stains  produced 
by  blood  containing  varying  percentages  of  haemoglobin.  In 
using  this  scale  a  drop  of  blood  is  allowed  to  fall  on  a  small 
section  of  filter  paper  and  the  resulting  color  is  compared  with 
the  ten  colors  of  the  scale.  When  the  color  in  the  scale  is 
found  which  corresponds  to  the  color  of  the  blood  stain  the 
accompanying  haemoglobin  value  is  read  off*  directly.  This 
is  a  very  convenient  method  for  determining  haemoglobin  at 
the  bedside.  There  is  a  possibility  of  the  colors  being  in- 
accurately printed,  however,  and  even  if  originally  correct  in 
tint,  under  the  continued  influence  of  air  and  light  they  must 
eventually  alter  somewhat. 

5.  Thoma-Zeiss  Haemocytometer. — This  is  an  instru- 
ment used  in  "blood  counting,"  i.  e.,  in  determining  the  num- 
ber of  erythrocytes  and  leucocytes.     The  instrument  consists 


Ill.nni). 


IM 


of  a  microscopic  slide  constructed  of  heavy  glass  and  provided 
with  a  central  counting  cell  (see  Fig.  70,  below).  This  cell, 
with  the  cover  glass  in  position,  is  exactly  0.1  millimeter  deep 

The  floor  of  the  cell  is  divided  by  delicate  lines  into  squares 
each  of  which  i>  4(1)0-  of  a  square  millimeter  in  area  (see  Fig, 
7_',  p.  [83  ).  The  volume  of  blood  therefore  between  any  par- 
ticular square  and  the  cover  glass  above  must  be    ,,/,,,,  cubic 


Thoma-Z  iixc;   Chamber.      (Da  Costa.) 

millimeter.  Accompanying  each  instrument  are  two  capil- 
lary pipettes  (Fig.  71,  p.  182),  each  constructed  with  a  mixing 
bulb  in  its  upper  portion.  Each  bulb  is  further  provided  with 
an  enclosed  glass  bead  which  is  of  great  assistance  in  mixing 
the  contents  of  the  chamber.  The  stem  of  each  pipette  is 
graduated  in  tenths  from  the  tip  to  the  bulb.  The  final  grad- 
uation at  the  upper  end  of  the  bulb  is  101  on  the  pipette  used 
in  mixing  the  blood  sample  in  which  the  erythrocytes  are 
counted  (erythrocytometer,  see  Fig.  71,  p.  182),  and  11  on  the 
pipette  used  in  mixing  the  blood  sample  for  the  leucocyte 
count  (leucocytometer, see  Fig.  71.  p.  182).  In  making  "blood 
counts"  with  the  haemocytometer  it  is  necessary  to  use  some 
diluting  fluid.  Two  very  satisfactory  forms  of  fluid  for  this 
purpose   are    Toison's   and    Sherrington's    solutions.1      When 


1  Toison's  solution  has  the  follow- 
ing formula  : 

Methyl  violet    0.025  gram. 

Sodium  chloride   1  gram. 

Sodium   sulphate    Sgrams. 

Glycerin    30  grams. 

Distilled  water  i6ograms. 


Sherrington's  solution  ha?  the  fol- 
lowing formula: 

Methylene-blue    0.1  gram. 

Sodium   chloride    1.2  gram. 

Neutral    potassium    ox- 
alate         1.2  gram. 

Distilled  water   300.0  grams. 


182 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  71. 


either  of  these  solutions  is  used  as  the  diluting  fluid  it  is  possi- 
ble to  make  a  very  satisfactory  count  of  both  the  erythrocytes 
and  leucocytes  from  the  same  preparation,  since  the  leucocytes 
are  stained  by  the  methyl  violet  or  methylene-blue. 

In  counting  the  erythrocytes  by  means 
of  the  hsemocytometer  proceed  as  follows : 
Thoroughly  cleanse  the  tip  of  the  finger 
or  lobe  of  the  ear  of  the  subject  by  the  use 
of  soap  and  water,  alcohol  and  ether  ap- 
plied in  the  sequence  just  given.  Punc- 
ture the  skin  by  means  of  a  needle  or  scalpel 
and  allow  the  blood  drop  to  form  without 
pressure.  Place  the  tip  of  the  pipette  in 
contact  with  the  blood  drop,  being  careful 
to  avoid  touching  the  skin,  and  draw  blood 
into  the  pipette  up  to  the  point  marked  0.5 
or  1  according  to  the  desired  dilution. 
Rapidly  wipe  the  tip  of  the  pipette  and 
immediately  fill  it  to  the  point  marked  101 
with  Toison's  or  Sherrington's  solution. 
Now  thoroughly  mix  the  blood  and  diluting 
fluid  within  the  mixing  chamber  by  tapping 
the  pipette  gently  against  the  finger  or  by 
shaking  it  while  held  securely  with  the 
thumb  at  one  end  and  the  middle  finger  at 
the  other.  After  the  two  fluids  have  been 
thoroughly  mixed  the  diluting  fluid  con- 
tained in  the  capillary-tube  below  the  bulb 
should  be  discarded  in  order  to  insure  the 
collection  of  a  drop  of  the  thoroughly  mixed 
blood  and  diluting  solution  for  examina- 
tion. Transfer  a  drop  from  the  pipette  to 
the  ruled  floor  of  the  counting  chamber  and, 
after   placing  the  cover-glass   firmly   in  position,1    allow   an 

1  If  the  cover  glass  is  in  accurate  apposition  to  the  counting  cell  New- 
ton's rings  may  be  plainly  observed. 


Thoma-Zeiss    Cap- 
illary  Pipettes. 
A,     Erythrocytom- 
eter ;   B,  leuco- 
cytometer. 


i:i.<  )(>!>. 


I83 


interval  of  a  few  minutes  to  elapse  For  the  corpuscles  to  settle 
before  making  the  count.  Now  place  the  slide  under  the 
microscope  and  count  the  number  of  erythrocytes  in  a  num- 
ber of  squares,  counting-  the  corpuscles  which  are  in  contact 
with  the  upper  and  the  righl  hand  boundaries  of  the  square 
as  belonging  to  that  square.    Take  the  squares  in  some  definite 

Fig.  72. 


Ordinary   Ruling   of   Thoma-Zeiss   Counting  Chamber.     (Da  Costa.) 

sequence  in  order  that  the  recounting  of  the  same  corpuscles 
may  be  avoided.  Of  course,  all  things  being  equal,  the 
greater  the  number  of  squares  examined  the  more  accurate 
the  count.  It  is  considered  essential  under  all  circumstances, 
where  an  accurate  count  is  desired,  that  the  counting  chamber 
shall  be  filled  at  least  twice  and  the  individual  counts  made  in 
each  instance,  as  indicated  above,  before  the  data  are  deemed 
satisfactory. 

To  calculate  the  number  of  erythrocytes  per  cubic  milli- 
meter of  undiluted  blood  proceed  as  follows :  Determine  the 
number  of  corpuscles  in  any  given  number  of  squares  and 
divide  this  total  by  the  number  of  squares,  thus  obtaining  the 
average  number  of  erythrocytes  per  square.  Multiply  this 
average  by  4,000  to  obtain  the  number  of  erythrocytes  per 


i84 


PHYSIOLOGICAL    CHEMISTRY. 


cubic  millimeter  of  diluted  blood,  and  multiply  this  product 
by  ioo  or  200,  according  to  the  dilution,  to  obtain  the  number 
of  erythrocytes  per  cubic  millimeter  of  undiluted  blood.    Thus  : 


Average  number  of  ery- 
throcytes per  square 


X  4,000  X  200  (or  100) 


Number  of  erythrocytes 
per  cubic  millimeter. 


Great  care  should  be  taken  to  see  that  the  capillary  pipette 
is  properly  cleaned.  After  using,  it  should  be  immediately 
rinsed  out  with  the  diluting  fluid,  then  with  water,  alcohol  and 
ether  in  the  sequence  given.  Finally  dry  air  should  be  drawn 
through  the  capillary  and  a  horse  hair  inserted  to  prevent  the 
entrance  of  dust  particles. 

In  counting  leucocytes  by  means  of  the  hsemocytometer  pro- 
ceed as  follows :  As  mentioned  above,  if  the  diluting  fluid  is 
either  Toison's  or  Sherrington's  solution  the  leucocytes  may 

Fig.  73. 


Zappert's    Modified    Ruling    of    Thoma-Zeiss  Counting    Chamber. 

Costa.) 


(Da 


be  counted  in  the  same  specimen  of  blood  in  which  the  ery- 
throcytes are  counted.  When  this  is  done  it  is  customary  to 
use  a  slide  provided  with  Zappert's  modified  ruling  (Fig.  73, 
above).     This  method  is  rather  more  accurate  than  the  older 


BLOOD.  is5 

one  of  counting  the  leucocytes  in  a  separate  specimen  of 
blood.  Furthermore  it  is  obviously  preferable  to  count  both 
the  erythrocytes   and   the   leucocytes    from   the   same   bl 

sample.  To  insure  accuracy  the  number  of  leucocytes  within 
the  whole  ruled  region  should  be  determined  in  duplicate  blood 
samples.     This  includes  the  examination  of  an  area  eighteen 

times  as  great  as  the  old  style  Thoma-Zeiss  central  ruling. 
This  region  then  would  correspond  to  3,600  of  the  small 
squares  and  if  duplicate  examinations  were  made  the  total 
number  of  small  squares  examined  would  aggregate  7.200. 

The  calculation  would  be  as  follows: 

Number  of  leucocytes  in  Number  of  leucocytes  per 

7.200  squares  X  200  X  4.000  -=-  7,200  =      cubic  millimeter 

[f  a  Zappert  slide  is  not  available  a  good  plan  to  follow  is 
to  place  a  diaphragm  in  the  tube  of  the  ocular  of  the  micro- 
scope consisting  of  a  circle  of  black  cardboard  or  metal1  hav- 
ing a  square  hole  in  its  centre  of  such  a  size  as  to  allow  of 
the  examination  of  exactly  100  squares  or  one-fourth  of  a 
square  millimeter  at  one  time.  With  this  arrangement  any 
portion  of  the  specimen  may  be  examined  and  counted  whether 
within  or  without  the  ruled  area.  In  counting  by  means  of 
this  device  it  is  of  course  helpful  if  the  microscope  is  pro- 
vided with  a  mechanical  stage,  but  even  without  this  arrange- 
ment, if  the  observer  is  careful  to  see  that  the  leucocytes  at 
the  extreme  boundary  of  one  field  move  to  the  opposite  bound- 
ary when  the  position  of  the  slide  is  changed,  the  device  may 
be  very  satisfactorily  employed.  The  leucocytes  should  be 
counted  in  36  of  the  diaphragm-fields  in  duplicate  specimens 
and  the  calculation  made  in  the  same  manner  as  explained 
above. 

If  the  leucocytes  are  counted  in  a  separate  specimen  of 
blood  ordinarily  the  diluting  fluid  is  0.3-0.5  per  cent  acetic 
acid,  a  fluid  in  which  the  leucocytes  alone  remain  visible. 
Under  these  conditions  the  dilution  is  customarily  made  in 
the  pipette  having  1 1  as  the  final  graduation.  The  capillary 
1  Ehrlich's  mechanical  eye-piece  is  also  very  satisfactory  for  this  purpose. 


i86 


PHYSIOLOGICAL    CHEMISTRY. 


portion  is  of  larger  caliber  and  so  requires  a  greater  amount  of 
blood  to  fill  it  to  the  0.5  or  1  mark  than  is  required  in  the  use 
of  the  other  form  of  pipette.  In  counting  the  leucocytes  ac- 
cording to  this  method  it  is  customary  to  draw  blood  into  the 
pipette  up  to  the  1  mark  and  immediately  fill  the  remaining 
portion  of  the  apparatus  to  the  11  graduation  with  the  0.3-0.5 
per  cent  acetic  acid.  It  then  remains  to  count  the  number  of 
leucocytes  in  the  whole  central  ruled  portion  of  400  squares. 
This  should  be  done  in  duplicate  samples  and  the  calculation 
made  as  follows : 

Number  of  leucocytes   in  Number  of  leucocytes  per 

800  squares.  X  ^00°  X  K>  -  800  =      cubk  mmimeten 


CI  I  \  PTER    XII. 
MILK. 

Milk  is  the  most  satisfactory  individual  food  material 
elaborated  by  nature.  It  contains  the  three  nutrients,  proteid, 
fat  and  carbohydrate  and  inorganic  salts  in  such  proportion 
as  to  render  it  a  very  acceptable  dietary  constituent.  It  is  a 
specific  product  of  the  secretory  activity  of  the  mammary 
gland.  It  contains,  as  the  principal  solids,  tri-olcin,  tri- 
palniitiii,  Iri-stcariti,  tri-butyrin,  caseinogen,  lactalbumin, 
lacto-globulin,  lactose  and  calcium  phosphate.  It  also  contains 
at  least  traces  of  lecithin,  cholesterin,  urea,  creatin,  creatinin 
and  the  tri-glycerides  of  caproic,  lauric  and  myristic  acids. 
Fresh  milk  is  amphoteric  in  reaction,  but  upon  standing  for  a 
sufficiently  long  time,  unsterilized,  it  becomes  acid  in  reaction, 
due  to  the  production  of  fermentation  lactic  acid, 

H      OH 

I         I 
H  -  C  -  C  -  COOH, 

H      H 

from  the  lactose  contained  in  it.  This  is  brought  about 
through  bacterial  activity.  The  white  color  is  imparted  to 
the  milk  partly  through  the  fine  emulsion  of  the  fat  and  partly 
through  the  medium  of  the  caseinogen  in  solution.  The 
specific  gravity  of  milk  varies  somewhat,  the  average  being 
about  1.030.     Its  freezing-point  is  about  —  0.560  C. 

Fresh  milk  does  not  coagulate  on  being  boiled  but  a  film  con- 
sisting of  a  combination  of  caseinogen  forms  on  the  surface. 
If  the  film  be  removed,  thus  allowing  a  fresh  surface  to  come 
in  contact  with  the  air,  a  new  film  will  form  indefinitely  upon 
the  application  of  heat.     Surface  evaporation  and  the  presence 

187 


i88 


PHYSIOLOGICAL    CHEMISTRY 


of  fat  facilitate  the  formation  of  the  film  but  are  not  essential. 
(  Rettger.)  If  the  milk  is  acid  in  reaction,  through  the  incep- 
tion of  lactic  acid  fermentation,  or  from  any  other  cause,  no 
film  will  form  when  heat  is  applied,  but  instead  a  true  coagula- 
tion will  occur.  The  milk-curdling  enzymes  of  the  gastric  and 
the  pancreatic  juice  have  the  power  of  splitting  the  caseinogen 
of  the  milk,  through  a  process  of  hydrolysis,  into  soluble  casein 


Fig.  74 


"• 


-    c 

I 


0 


- 


w    v     w  /-> 
b 

Normal  Milk  and  Colostrum. 
a,  Normal  milk  ;  b,  Colostrum. 

and  a  peptone-like  body.  This  soluble  casein  then  forms  a 
combination  with  the  calcium  of  the  milk  and  an  insoluble  curd 
of  calcium  casein  or  casein  results.  The  clear  fluid  surround- 
ing the  curd  is  known  as  whey. 

The  most  pronounced  difference  between  human  milk  and 
cow's  milk  is  in  the  proteid  content,  although  there  are  also 
differences  in  the  fats  and  likewise  striking  biological  dif- 
ferences difficult  to  define  chemically.  It  lias  been  shown  that 
the  caseinogen  of  human  milk  differs  from  the  caseinogen  of 
cow's  milk  in  being  more  difficult  to  precipitate  by  acid  or 
coagulate  by  rennin.  The  casein  curd  also  forms  in  a  much 
looser  and  more  flocculent  manner  than  that  from  cow's  milk 
and  is   for  this  reason  much  more  easily  digested  than  the 


MILK. 


189 


latter.  Interesting  data  relative  to  the  composition  of  milk 
from  various  sources,  may  be  gathered  from  the  following 
table  which  was  compiled  mainly  from  the  results  of  investi- 
gations by  Bunge  and  by  Abderhalden.  It  will  be  noted  that 
the  composition  of  the  milk  varies  directly  with  the  length  of 
time  needed  for  the  young  of  the  particular  species  to  double 
in  weight. 


Peiiod  in  which 

weight  of  the 

new-born  is 

doubled  (days). 

ISO 
60 

47 
22 

15 

•4 
9-5 
9 
6 

100  parts  of  milk  contain 

Species. 

•  ids. 

..6 
2.0 
3-5 
3-7 
4-9 
5-2 
7.0 

7-4 
10.4 

Salts 

0.2 
0.4 
0.7 
o.S 
0.8 
0.8 
1.0 
i-3 

2-5 

Calcium. 

O.O33 
O.I24 
O.  160 
O.I97 
O.245 
O.249 

o-455 
0.891 

Phosphoric 
acid 

M;m 

Cow 



Pig 



D°g 

Rabbit 

O.O47 
O.I3I 

O.I97 
O.284 
O.293 
O.308 

O.508 
O.997 

Lactose,  the  carbohydrate  constituent  of  milk,  is  an  impor- 
tant member  of  the  disaccharide  group.      It  occurs  only  in 

Fig.  75. 


Lactose. 


milk,  except  as  it  is  found  in  the  urine  of  women  during  preg- 
nancy, during  the  nursing  period  and  sbon  after  weaning;  it  also 


19°  PHYSIOLOGICAL    CHEMISTRY. 

occurs  in  the  urine  of  normal  persons  after  the  ingestion  of  a 
very  large  amount  of  lactose  in  the  food.  It  is  not  derived 
directly  from  the  blood  but  is  a  specific  product  of  the  cellular 
activity  of  the  mammary  gland.  It  has  strong  reducing  power, 
is  dextro-rotatory  and  forms  an  osazone  with  phenylhydrazin. 
The  souring  of  milk  is  due  to  the  formation  of  lactic  acid 
from  lactose  through  the  agency  of  the  bacterium  lactis. 
Putrefactive  bacteria  in  the  alimentary  canal  may  bring  about 
this  same  reaction.     Lactose  is  not  fermentable  by  pure  yeast. 

Caseinogen,  the  principal  proteid  constituent  of  milk  be- 
longs to  the  group  of  phospho-proteids.  It  has  acidic  properties 
and  combines  with  bases  to  produce  salts.  It  is  not  coagulable 
upon  boiling  and  is  precipitated  from  its  neutral  solution  by 
certain  metallic  salts  as  well  as  upon  saturation  with  sodium 
chloride  or  magnesium  sulphate.  Its  acid  solution  is  precipi- 
tated by  an  excess  of  mineral  acid. 

Lactalbumin  and  lacto-globulin,  the  other  proteid  consti- 
tuents of  milk,  closely  resemble  serum  albumin  and  serum 
globulin  in  their  general  properties. 

Colostrum  is  the  name  given  to  the  product  of  the  mam- 
mary gland  secreted  for  a  short  time  before  parturition  and 
during  the  early  period  of  lactation  (see  Fig.  74,  p.  188).  It 
is  yellowish  in  color,  contains  more  solid  matter  than  ordinary 
milk  and  has  a  higher  specific  gravity  (1. 040-1. 080).  The 
most  striking  difference  between  colostrum  and  ordinary  milk 
is  the  high  percentage  of  lactalbumin  and  lactoglobulin  in  the 
former.  This  abnormality  in  the  proteid  content  is  respon- 
sible for  the  coagulation  of  colostrum  upon  boiling. 

Experiments  on  Milk. 

1.  Reaction. — Test  the  reaction  of  fresh  cow's  milk  to 
litmus. 

2.  Biuret  Test. — Make  the  biuret  test  according  to  direc- 
tions given  on  page  45. 

3.  Microscopical  Examination. — Examine  fresh  whole 
milk,  skimmed  or  centrif u gated  milk  and  colostrum  under  the 


MILK.  I9I 

microscope.     Compare  the  microscopical  appearance  with  Fig. 
74,  page  188. 

4.  Specific  Gravity. — Determine  the  specific  gravity  of 
both  whole  and  skimmed  milk.  Which  possesses  the  higher 
specific  gravity?  Explain  why  this  is  so. 

5.  Film  Formation. — Place  10  c.c.  of  milk  in  a  small 
beaker  and  boil  a  few  minutes.  Note  the  formation  of  a  film. 
Remove  the  film  and  heat  again.  Does  the  film  now  form? 
Of  what  substance  is  this  film  composed?  The  biuret  test  was 
positive,  why  do  we  not  get  a  coagulation  here  when  we  heat 
to  boiling? 

6.  Coagulation  Test. — Place  about  5  c.c.  of  milk  in  a  test- 
tube,  acidify  slightly  with  dilute  acetic  acid  and  heat  to  boil- 
ing.    Do  you  get  any  coagulation?    Why? 

7.  Action  of  Hot  KOH. — To  a  little  milk  in  a  test-tube 
add  a  few  drops  of  KOH  and  heat.  A  yellow  color  develops 
and  gradually  deepens  into  a  brown.  To  what  is  the  formation 
of  this  color  due? 

8.  Test  for  Chlorides. — To  about  5  c.c.  of  milk  in  a  test- 
tude  add  a  few  drops  of  very  dilute  nitric  acid  to  form  a  pre- 
cipitate. Filter  off  this  precipitate  and  test  the  filtrate  for 
chlorides.     Does  milk  contain  any  chlorides? 

9.  Guaiac  Test. — To  about  5  c.c.  of  water  in  a  test-tube 
add  3  drops  of  milk  and  enough  alcoholic  solution  of  guaiac 
(strength  about  1:6c)1  to  cause  a  turbidity.  Thoroughly 
mix  the  fluids  by  shaking  and  observe  any  change  which  may 
gradually  take  place  in  the  color  of  the  mixture.  If  no  blue 
color  appears  in  a  short  time,  heat  the  tube  gently  below 
6o°  C.  and  observe  whether  the  color  reaction  is  hastened. 
In  case  a  blue  color  does  not  appear  in  the  course  of  a  few 
minutes,  add  hydrogen  peroxide  or  old  turpentine,  drop  by 
drop,  until  the  color  is  observed.  Fresh  milk  will  frequently 
give  this  blue  color  when  treated  with  an  alcoholic  solution  of 

1  Buckmaster  advises  the  use  of  an  alcoholic  solution  of  guaiaconic 
acid  instead  of  an  alcoholic  solution  of  guaiac  resin.  Guaiaconic  acid  is 
a  constituent  of  guaiac  resin. 


I92  PHYSIOLOGICAL    CHEMISTRY. 

guaiac  without  the  addition  of  hydrogen  peroxide  or  old  tur- 
pentine.    See  discussion  on  page  158. 

10.  Saturation  with  MgS04. — Place  about  5  c.c.  of  milk 
in  a  test-tube  and  saturate  with  solid  magnesium  sulphate. 
What  is  this  precipitate? 

11.  Influence  of  Rennin  on  Milk. — Prepare  a  series  of 
five  tubes  as  follows  : 

(a)  5  c.c.  of  fresh  milk  -f  0.2  per  cent  HC1  (add  drop  by 
drop  until  a  precipitate  forms). 

(b)  5  c.c.  of  fresh  milk  +  5  drops  of  rennin  solution. 

(c)  5  c.c.  of  fresh  milk  +  10  drops  of  0.5  per  cent  Na2C03. 

(d)  5  c.c.  of  fresh  milk  -|-  10  drops  of  ammonium  oxalate. 

(e)  5  c.c.  of  fresh  milk  -f  5  drops  of  0.2  per  cent  HC1. 
Xow  to  each  of  the  tubes  (c),  (d)  and  (e)  add  5  drops  of 

rennin  solution.  Place  the  whole  series  of  five  tubes  at  400  C. 
and  after  10-15  minutes  note  what  is  occurring  in  the  differ- 
ent tubes.     Give  a  reason  for  each  particular  result. 

12.  Preparation  of  Caseinogen. — Fill  a  large  beaker  one- 
third  full  of  skimmed  (or  centrifugated)  milk  and  dilute  it 
with  an  equal  volume  of  water.  Add  dilute  hydrochloric  acid 
until  a  flocculent  precipitate  forms.  Stir  after  each  acidifica- 
tion and  do  not  add  an  excess  of  the  acid  as  the  precipitate 
would  dissolve.  Allow  the  precipitate  to  settle,  decant  the 
supernatant  fluid  and  reserve  it  for  use  in  later  (13-15)  ex- 
periments. Filter  off  the  precipitate  of  caseinogen  and  remove 
the  excess  of  moisture  by  pressing  it  between  filter  papers. 
Transfer  the  caseinogen  to  a  small  beaker,  add  enough  95 
per  cent  alcohol  to  cover  it  and  stir  for  a  few  moments. 
Filter,  and  press  the  precipitate  between  filter  papers  to  remove 
the  alcohol.  Transfer  the  caseinogen  again  to  a  small  dry 
beaker,  cover  the  precipitate  with  ether  and  heat  on  a  water- 
bath  for  ten  minutes,  stirring  continuously.  Filter  (reserve 
the  filtrate),  and  press  the  precipitate  as  dry  as  possible  be- 
tween filter  papers.  Open  the  papers  and  allow  the  ether  to 
evaporate  spontaneously.  Grind  the  precipitate  to  a  powder 
in  a  mortar.  Upon  the  caseinogen  prepared  in  this  way  make 
the  following  tests: 


MILK.  193 

(a)  Solubility. — Try  the  solubility  ill  the  ordinary  solvents 

(b)  M Moris  Reaction. — .Make  the  test  according  to  the 
directions  given  on  page  44. 

1  c)  Biuret  Test. — Make-  the  test  according  to  the  directions 
given  on  page  45. 

i  d 1  Xanthoproteic  Reaction. — Make  the  test  according  to 
the  directions  given  on  page  44. 

(e)  Loosely  Combined  Sulphur. — Test  for  loosely  com- 
bined sulphur  according  to  the  directions  given  on  page  52. 

(/)  Fusion  Test  for  Phosphorus. — Test  for  phosphorus  by 
fusion  according  to  directions  given  on  page  223. 

13.  Coagulable  Proteids  of  Milk. — Place  the  filtrate  from 
the  original  caseinogen  precipitate  in  a  casserole  and  heat,  on 
a  wire  gauze,  over  a  free  flame.  As  the  solution  concentrates, 
a  coagulum  consisting  of  lactalbumin  and  lactoglobulin  will 
form.  Continue  to  concentrate  the  solution  until  the  volume 
is  about  one-half  that  of  the  original  solution.  Filter  off  the 
coagulable  proteids  (reserve  the  filtrate)  and  test  them  as 
follows : 

(a)  Milloris  Reaction. — Make  the  test  according  to  the 
directions  given  on  page  44. 

(b)  Biuret  Test. — Make  the  test  according  to  the  direc- 
tions given  on  page  45. 

(c)  Xanthoproteic  Reaction. — Make  the  test  according  to 
the  directions  given  on  page  44. 

14.  Detection  of  Calcium  Phos- 
phate.— Evaporate  the  filtrate  from  the 
coagulable  proteids,  on  a  water-bath, 
until  crystals  begin  to  form.  It  may 
be  necessary  to  concentrate  to  15  c.c. 
before  any  crystallization  will  be  ob- 
served. Cool  the  solution,  filter  oft  the 
crystals  (reserve  the  filtrate)  and  test  '  calch-m  Phosphate. 
them  as  follows : 

(a)   Microscopical  Examination. — Examine  the  crystals  and 
compare  them  with  those  in  Fig.  76,  above. 
14 


194  PHYSIOLOGICAL    CHEMISTRY. 

(b)  Dissolve  the  crystals  in  nitric  acid.  Test  part  of  the 
acid  solution  for  phosphates.  Render  the  remainder  of  the 
solution  slightly  alkaline  with  ammonia,  then  acidify  with  acetic 
acid  and  add  ammonium  oxalate.  Examine  the  crystals  under 
the  microscope  and  compare  them  with  those  in  Fig.  99,  p.  320. 

15.  Detection  of  Lactose. — Concentrate  the  filtrate  from 
the  calcium  phosphate  until  it  is  of  a  syrup-like  consistency. 
Allow  it  to  stand  over  night  and  observe  the  formation  of 
crystals  of  lactose.     Make  the  following  experiments : 

(a)  Microscopical  Examination. — Examine  the  crystals  and 
compare  them  with  those  in  Fig.  75,  page  189. 

(&)  Fehlings  Test. — Try  Fehling's  test  upon  the  mother 
liquor. 

(c)  Phenylhydrazin  Test. — Apply  the  phenylhydrazin  test 
to  some  of  the  mother  liquor  according  to  the  directions  given 
on  page  5. 

16.  Milk  Fat. —  (a)  Evaporate  the  ether  filtrate  from  the 
caseinogen  (Experiment  12)  and  observe  the  fatty  residue. 
The  milk  fat  was  carried  down  with  the  precipitate  of  case- 
inogen and  was  removed  when  the  latter  was  treated  with 
ether.  If  centrifugated  milk  was  used  in  the  preparation  of 
the  caseinogen  the  amount  of  fat  in  the  ether  filtrate  may  be 
very  small.  To  secure  a  larger  yield  of  fat  proceed  accord- 
ing to  directions  given  under  (b)  below. 

(b)  To  25  c.c.  of  whole  milk  in  an  evaporating  dish  add  a 
little  sand  or  filter  paper  and  evaporate  the  fluid  to  dryness 
on  a  water-bath.  Grind  or  break  up  the  residue  after  cooling 
and  extract  with  ether  in  a  flask.  Filter  and  remove  the  ether 
from  the  filtrate  by  evaporation.  How  can  you  identify  fats 
in  the  ethereal  residue? 

17.  Saponification  of  Butter. — Dissolve  a  small  amount  of 
butter  in  alcohol  made  strongly  alkaline  with  potassium  hy- 
droxide. Place  the  alcoholic-potash  solution  in  a  casserole, 
add  about  100  c.c.  of  water  and  boil  for  10-15  minutes  or 
until  the  odor  of  alcohol  cannot  be  detected.  Place  the  cas- 
serole in  a  hood  and  neutralize  the  solution  with   sulphuric 


MILK.  195 

acid.     Note  the  odor  of  volatile  fatty  acids  particularly  butyric 
acid. 

[8.   Detection  of  Preservatives. —  (a)    Formaldehyde. 

I.  Gallic  Acid  Test.—  Acidify  30  c.c.  of  milk  with  2  c.c.  of 
normal  sulphuric  acid  and  distil.  Add  <>._'  0.3  c.c.  of  a  satu- 
rated alcoholic  solution  of  gallic  acid  to  the  first  5  c.c  of  the 
distillate,  then  incline  the  test-tube  and  slowly  introduce  3  5 
c.c.  of  concentrated  sulphuric  acid,  allowing  it  to  run  slowly 
d<  i\\  n  the  side  of  the  tube.  A  green  ring,  which  finally  changes 
to  blue,  is  formed  at  the  juncture  of  the  fluids.  This  is 
claimed,  by  Sherman,  to  he  twice  as  delicate  as  either  the  sul- 
phuric acid  or  the  hydrochloric  acid  test  for  formaldehyde. 

II.  Hydrochloric  Acid  Test. — Mix  10  c.c.  of  milk  and  10  c.c. 
of  concentrated  hydrochloric  acid  containing  about  0.002  gram 
of  ferric  chloride  in  a  small  porcelain  evaporating  dish  or 
casserole  and  gradually  raise  the  temperature  of  the  mixture 
nearly  to  the  boiling-point,  with  occasional  stirring.  If 
formaldehyde  is  present  a  violet  color  is  produced,  while  a 
brown  color  develops  in  the  absence  of  formaldehyde.  In  case 
of  doubt  the  mixture,  after  having  been  heated  nearly  to  the 
boiling-point  for  about  one  minute,  should  be  diluted  with 
50-75  c.c.  of  water,  and  the  color  of  the  diluted  fluid  carefully 
noted,  since  the  violet  color  if  present  will  quickly  disappear 
Formaldehyde  may  be  detected  by  this  test  when  present  in  the 
proportion  1  :  250,000. 

(b)  Salicylic  Acid  and  Salicylates. — Remont's  Method.1 
Acidify  20  c.c.  of  milk  with  sulphuric  acid,  shake  well  to 
break  up  the  curd,  add  25  c.c.  of  ether,  mix  thoroughly 
and  allow  the  mixture  to  stand.  By  means  of  a  pipette  remove 
5  c.c.  of  the  ethereal  extract,  evaporate  it  to  dryness,  boil  the 
residue  with  10  c.c.  of  40  per  cent  alcohol  and  cool  the  alcohlic 
solution.  Make  the  volume  10  c.c,  filter  through  a  dry  paper 
if  necessary  to  remove  fat,  and  to  5  c.c.  of  the  filtrate,  which 
represents  2  c.c.  of  milk,  add  2  c.c.  of  a  2  per  cent  solution  of 
ferric  chloride.  The  production  of  a  purple  or  violet  color  indi- 
cates the  presence  of  salicylic  acid. 

1  Sherman's  Organic  Analysis,  p.  232. 


I96  PHYSIOLOGICAL    CHEMISTRY. 

This  test  may  form  the  basis  of  a  quantitative  method  by 
diluting  the  final  solution  to  50  c.c.  and  comparing  this  with 
standard  solutions  of  salicylic  acid.  The  colorimetric  com- 
parisons may  be  made  in  a  Duboscq  colorimeter. 

(c)  Hydrogen  Peroxide. — Add  2-3  drops  of  a  2  per  cent 
aqueous  solution  of  paraphenylenediamine  hydrochloride  to 
10-15  c.c.  of  milk.  If  hydrogen  peroxide  is  present  a  blue 
color  will  be  produced  immediately  upon  shaking  the  mixture 
or  after  allowing  it  to  stand  for  a  few  minutes.  It  is  claimed 
that  hydrogen  peroxide  may  be  detected  by  this  test  when 
present  in  the  proportion  1 :  40,000. 

(d)  Boric  Acid  and  Borates. — To  the  ash,  obtained  accord- 
ing to  the  directions  given  on  p.  — ,  add  2  drops  of  dilute 
hydrochloric  acid  and  1  c.c.  of  water.  Place  a  strip  of  turmeric 
paper  in  the  dish  and  after  allowing  it  to  soak  for  about  one 
minute  remove  it  and  allow  it  to  dry  in  the  air.  The  pres- 
ence of  boric  acid  is  indicated  by  the  production  of  a  deep  red 
color  which  changes  to  green  or  blue  upon  treatment  with  a 
dilute  alkali.  This  test  is  supposed  to  show  boric  acid  when 
present  in  the  proportion  1  :  8000. 


CHAPTER    XIII. 

EPITHELIAL   AND    CONNECTIVE   TISSUES. 

EPITHELIAL    TISSUE   (KERATIN). 

The  albuminoid  keratin  constitutes  the  major  portion  of 
hair,  horn,  hoof,  feathers,  nails  and  the  epidermal  layer  of 
the  skin.  There  is  a  group  of  keratins  the  members  of  which 
possess  very  similar  properties.  The  keratins  as  a  group  are 
insoluble  in  the  usual  proteid  solvents  and  are  not  acted  upon 
by  the  gastric  or  pancreatic  juices.  They  all  respond  to  the 
xanthoproteic  and  Millon  reactions  and  are  characterized  by 
containing  large  amounts  of  sulphur.  Keratin  from  any  of 
its  sources  may  be  prepared  in  a  pure  form  by  treatment,  in 
sequence,  with  artificial  gastric  juice,  artificial  pancreatic  juice, 
boiling  alcohol  and  boiling  ether,  from  twenty- four  to  forty- 
eight  hours  being  devoted  to  each  process. 

Experiments  ox  Epithelial  Tissri:. 
Keratin. 

Horn  shavings  may  be  used  in  the  experiments  which  fol- 
low: 

i.  Solubility. — Test  the  solubility  of  keratin  in  the  ordinary 
solvents  (see  p.  4). 

2.  Mill 011's  Reaction. 

3.  Xanthoproteic  Reaction. 

4.  Adamkiewictfs  Reaction. 

5.  Iloplcius-Colc  Reaction. 

6.  Test  for  Loosely  Combined  Sulphur. 

CONNECTIVE  TISSUE. 
I.     WHITE  FIBROUS  TISSUE. 


The  principal  solid  constituent  of  white  fibrous  connective 

^en. 
i97 


tis>ue  is  the  albuminoid  collagen.     This  body  is  also  found  in 


198  PHYSIOLOGICAL    CHEMISTRY. 

smaller  percentage  in  cartilage,  bone  and  ligament,  but  the 
collagen  from  the  various  sources  is  not  identical  in  composi- 
tion. In  common  with  the  keratins,  collagen  is  insoluble  in 
the  usual  proteid  solvents.  It  differs  from  keratin  in  con- 
taining less  sulphur.  One  of  the  chief  characteristics  of  col- 
lagen is  the  property  of  being  hydrolyzed  by  boiling  acid  or 
water  with  the  formation  of  gelatin.  It  gives  Millon's  re- 
action as  well  as  the  xanthoproteic  and  biuret  tests. 

The  form  of  white  fibrous  tissue  most  satisfactory  for  gen- 
eral experiments  is  the  tendo  Achillis  of  the  ox.  According 
to  Buerger  and  Gies  the  fresh  tissue  has  the  following  com- 
position : 

Water   62.87% 

Solids    37-13 

Inorganic  matter  0.47 

Organic  matter 36.66 

Fatty  substance   (ether-soluble)  . .  . , 1.04 

Coagulable  proteid    0.22 

Mucoid    1.28 

Elastin    1.63 

Collagen    31.59 

Extractives,  etc 0.90 

The  mucoid  mentioned  above  is  called  tendomucoid  and  is 
a  glucoproteid.  It  possesses  properties  similar  to  those  of 
other  connective  tissue  mucoids,  e.  g.,  osseomucoid  and  chon- 
dromucoid. 

Gelatin,  the  body  which  results  from  the  hydrolysis  of  col- 
lagen, is  also  an  albuminoid.  It  responds  to  nearly  all  the  pro- 
teid tests.  It  differs  from  the  keratins  and  collagen  in  being 
easily  digested  and  absorbed.  Gelatin  is  not  a  satisfactory 
substitute  for  the  proteid  constituents  of  a  normal  diet  how- 
ever, since  a  certain  portion  of  its  nitrogen  is  not  available 
for  the  uses  of  the  organism.  Gelatin  from  cartilage  differs 
from  the  gelatin  from  other  sources  in  containing  a  lower 
percentage  of  nitrogen. 

Experiments  on  White  Fibrous  Tissue. 
The  tendo  Achillis  of  the  ox  may  be  taken  as  a  satisfactory 
type  of  the  white  fibrous  connective  tissue. 


EPITHELIAL    AND    CONNECTIVE    TISSUES.  I99 

i.  Preparation  of  Tendomucoid. — Dissect  away  the  fascia 
from  about  the  tendon  and  cut  the  clean  tendon  into  small 
pieces.  Wash  the  pieces  in  water,  changing  the  wash  water 
often  in  order  to  remove  as  much  as  possible  of  the  soluble 
proteid  and  inorganic  salts.  Transfer  the  washed  pieces  of 
tendon  to  a  flask  and  add  300  c.c.  of  lialf-su titrated  lime-water.1 
Shake  the-flask  at  intervals  for  twenty-four  hours.  Filter  off 
the  pieces  of  tendon  and  precipitate  the  mucoid  with  dilute 
hydrochloric  acid.  Allow  the  mucoid  precipitate  to  settle, 
decant  the  supernatant  fluid  and  filter  the  remainder.  Test 
the  mucoid  as  follows : 

(a)  Solubility. — Try  the  solubility  in  the  ordinary  solvents 
(see  p.  4). 

(b)  Biuret  Test. — First  dissolve  the  mucoid  in  KOH  solu- 
tion and  then  add  a  dilute  solution  of  CuS04. 

(c)  Test  for  Loosely  Combined  Sulphur. 

(d)  Hydrolysis  of  Tendomucoid. — Place  the  remainder 
of  the  mucoid  in  a  small  beaker,  add  about  30  c.c.  of  water 
and  2  c.c.  of  dilute  hydrochloric  acid  and  boil  until  the  solu- 
tion becomes  dark  brown.  Cool  the  solution,  neutralize  it  with 
solid  KOH  and  test  by  Fehling's  test.  With  a  reduction 
of  Fehling's  solution  and  a  positive  biuret  test  what  do  you 
conclude  regarding  the  nature  of  tendomucoid  ? 

2.  Collagen. — This  substance  is  present  in  the  tendon  to 
the  extent  of  about  32  per  cent.  Therefore  in  making  the  fol- 
lowing tests  upon  the  pieces  of  tendon  from  which  the  mucoid, 
soluble  proteid  and  inorganic  salts  were  removed  in  the  last 
experiment,  we  may  consider  the  tests  as  being  made  upon 
collagen. 

(a)  Solubility. — Cut  the  collagen  into  very  fine  pieces  and 
try  its  solubility  in  the  ordinary  solvents  (see  page  4). 

(b)  Millon's  Reaction. 

(c)  Biuret  Test. 

(d )  Xanthoproteic  Reaction. 

1  Made  by  mixing  equal  volumes  of  saturated  lime-water  and  water  from 
the  faucet. 


200  PHYSIOLOGICAL    CHEMISTRY. 

(e)  Hopkins-Cole  Reaction. 

(/)  Test  for  Loosely  Combined  Sulphur. — Take  a  large 
piece  of  collagen  in  a  test-tube  and  add  about  5  c.c.  of  KOH 
solution.  Heat  until  the  collagen  is  partly  decomposed,  then 
add  1-2  drops  of  plumbic  acetate  and  again  heat  to  boiling. 

(g)  Hydrolysis  of  Collagen. — Transfer  the  remainder  of 
the  pieces  of  collagen  to  a  casserole,  fill  the  vessel  about  two- 
thirds  full  of  water  and  boil  for  several  hours,  adding  water  at 
intervals  as  needed.  By  this  means  the  collagen  is  hydrolyzed 
and  a  body  known  as  gelatin  is  formed. 

3.  Gelatin. — On  the  gelatin  formed  from  the  hydrolysis 
of  collagen  in  the  above  experiment  (g),  or  on  gelatin  fur- 
nished by  the  instructor  make  the  following  tests : 

(a)  Solubility. — Try  the  solubility  in  the  ordinary  solvents 
(see  page  4)  and  in  hot  water. 

(b)  Milton's  Reaction. 

(c)  Hopkins-Cole  Reaction. — Conduct  this  test  according  to 
the  modification  given  on  page  51. 

(d)  Test  for  Loosely  Combined  Sulphur. 

Make  the  following  tests  upon  a  solution  of  gelatin  in  hot 
water : 

(a)  Precipitation  by  Mineral  Acids. — Is  it  precipitated  by 
strong  mineral  acids  such  as  concentrated  hydrochloric  acid? 

(b)  Salting-Out  Experiment. — Saturate  a  little  of  the  solu- 
tion with  solid  ammonium  sulphate.  Is  the  gelatin  precip- 
itated? Repeat  the  experiment  with  sodium  chloride.  What 
is  the  result? 

(c)  Precipitation  by  Metallic  Salts. — Is  it  precipitated  by 
metallic  salts  such  as  cupric  sulphate,  mercuric  chloride  and 
plumbic  acetate? 

(d)  Coagulation  Test. — Does  it  coagulate  upon  boiling? 

(e)  Precipitation  by  Alkaloidal  Reagents. — Is  it  precipi- 
tated by  such  reagents  as  picric  acid,  tannic  acid  and  trichlor- 
acetic acid? 

(/)   Biuret  Test. — Does  it  respond  to  the  biuret  test? 


EPITHELIAL    AND    CONNECTIVE     riSSUES.  201 

(g)  Precipitation  by  Alcohol. — Fill  a  test-tube  one-half 
full  of  95  per  cent  alcohol  and  pour  in  a  small  amount  of  con- 
centrated gelatin  solution.  Do  you  get  a  precipitate?  How 
would  you  prepare  pure  gelatin  from  the  tendo  Achillis  of 
the  ox  ? 

[I.     YELLOW  ELASTIC  TISSUE  (ELASTIN). 

The  Ligatnentum  michcr  of  the  ox  may  be  taken  as  a  satis- 
factory type  of  the  yellow  elastic  connective  tissue.  The 
principal  solid  constituent  of  this  tissue  is  elastin,  a  member  of 

the  albuminoid  group.  In  common  with  the  keratins  and 
collagen,  elastin  is  an  insoluble  body  and  gives  the  proteid 
color  reactions.  It  differs  from  keratin  principally  in  the  fact 
that  it  may  be  digested  by  enzymes  and  that  it  contains  a  very 
small  amount  of  sulphur. 

Yellow  elastic  tissue  also  contains  mucoid  and  collagen  but 
these  are  present  in  much  smaller  amount  than  in  white  fibrous 
tissue,  as  may  be  seen  from  the  following  percentage  composi- 
tion of  the  fresh  ligamentum  nucha:  of  the  ox  as  determined 
by  Vandegrift  and  Gies  : 

Water   57-57% 

Solids    4243 

Inorganic  matter  0.47 

Organic  matter    41 .96 

Fatty  substance   (ether-soluble) 1.12 

Coagulable  proteid    0.62 

Mucoid    0.53 

Elastin     31.67 

Collagen    7.23 

Extractives,  etc 080 

Experiments  ox  Elastin. 
1.  Preparation  of  Elastin  (Richards  and  Gies). — Cut  the 
ligament  into  fine  strips,  run  it  through  a  meat  chopper  and 
wash  the  finely  divided  material  in  cold,  running  water  for 
24-48  hours.  Add  an  excess  of  half-saturated  lime-water 
(see  note  at  bottom  of  p.  199)  and  allow  the  hashed  ligament 


202  PHYSIOLOGICAL    CHEMISTRY. 

to  extract  for  48-72  hours.  Decant  the  lime-water,  remove 
all  traces  of  alkali  by  washing  in  water  and  then  boil  in  water 
with  repeated  renewals  until  only  traces  of  proteid  material 
can  be  detected  in  the  wash  water.  Decant  the  fluid  and  boil 
the  ligament  in  10  per  cent  acetic  acid  for  a  few  hours.  Treat 
the  pieces  with  5  per  cent  hydrochloric  acid  at  room  temper- 
ature for  a  similar  period,  extract  again  in  hot  acetic  acid  and 
in  cold  hydrochloric  acid.  Wash  out  traces  of  acid  by  means 
of  water  and  then  thoroughly  dehydrolyze  by  boiling  alcohol 
and  boiling  ether  in  turn.  Dry  in  an  air-bath  and  grind  to  a 
powder  in  a  mortar. 

2.  Solubility. — Try  the  solubility  of  the  finely  divided 
elastin,  prepared  by  yourself  or  furnished  by  the  instructor, 
in  the  ordinary  solvents  (see  page  4).  How  does  its  solu- 
bility compare  with  that  of  collagen? 

3.  Millon's  Reaction. 

4.  Xanthoproteic  Reaction. 

5.  Biuret  Test. 

6.  Hopkins-Cole  Reaction. — Conduct  this  test  according 
to  the  modification  given  on  page  5 1 . 

7.  Test  for  Loosely  Combined  Sulphur. 

III.     CARTILAGE. 

The  principal  solid  constituents  of  the  matrix  of  cartilagi- 
nous tissue  are  chondro mucoid,  chondroitin-sulphuric  acid, 
chondroalbumoid  and  collagen.  Chondromucoid  differs  from 
the  mucoids  isolated  from  other  connective  tissues  in  the  large 
amount  of  chondroitin-sulphuric  acid  obtained  upon  decom- 
position. Besides  being  an  important  constituent  of  all  forms 
of  cartilage,  chondroitin-sulphuric  acid  has  been  found  in 
bone,  ligament,  the  mucosa  of  the  pig's  stomach,  the  kidney  of 
the  ox,  the  inner  coats  of  large  arteries  and  in  human  urine. 
It  may  be  decomposed  through  the  action  of  acid  and  yields 
a  nitrogenous  body  known  as  chondroitin  and  later  this  body 
yields  chondrosin.  Chondrosin  is  also  a  nitrogenous  body  and 
has  the  power  of  reducing  Fehling's  solution  more  strongly 


EPITHELIAL    AND    CONNECTIVE   TISSUES.  203 

than  dextrose.  Sulphuric  acid  is  a  by-product  in  the  forma- 
tion of  chondroitin.  and  acetic  arid  is  a  by-product  in  the  for- 
mation of  chondrosin. 

Chondroalbumoid  is  similar  in  some  respects  to  elastin  and 
keratin.  It  differs  from  keratin  in  being  soluble  in  gastric 
juice  and  in  containing  considerably  less  sulphur  than  any 
member  of  the  keratin  group.     It  gives  the  usual  proteid  color 

reactions. 

Experiments  on  Cartilage. 

1.  Preparation  of  the  Cartilage. — Boil  the  trachea  of  an 
ox  in  water  until  the  cartilage  rings  may  be  completely  freed 
frpm  the  surrounding  tissue.  Use  the  cartilage  so  obtained 
in  the  following  experiments. 

_'.  Solubility. — Cut  one  of  the  rings  into  very  small  pieces 
and  try  the  solubility  of  the  cartilage  in  the  ordinary  solvents 
(see  page  4). 

3.  Millon's  Reaction. 

4.  Xanthoproteic  Reaction. 

5.  Hopkins-Cole  Reaction. — Conduct  this  test  according  to 
the  modification  given  on  page  51. 

6.  Test  for  Loosely  Combined  Sulphur. 

7.  Preparation  of  Cartilage  Gelatin. — Cut  the  remaining 
cartilage  rings  into  small  pieces,  place  them  in  a  casserole  with 
water  and  boil  for  several  hours.  Filter  while  the  solution  is 
still  hot.  Observe  that  the  filtrate  soon  becomes  more  or  less 
solid.  What  is  the  reason  for  this?  Bring  a  portion  of  the 
material  into  solution  by  heat  and  try  the  following  tests : 

(a)  Biuret  Test. 

(b)  Test  for  Loosely  Combined  Sulphur. 

(c)  To  about  5  c.c.  of  the  solution  in  a  test-tube  add  a 
few  drops  of  barium  chloride.  Do  you  get  a  precipitate,  and 
if  so  to  what  is  the  precipitate  due? 

(d)  To  about  5  c.c.  of  the  solution  in  a  test-tube  add  a  few 
drops  of  dilute  hydrochloric  acid  and  boil  for  a  few  moments. 
Now  add  a  little  barium  chloride  to  this  solution.  Is  the  pre- 
cipitate any  larger  than  that  obtained  in  the  preceding  experi- 
ment ?     Why  ? 


204  PHYSIOLOGICAL    CHEMISTRY. 

(e)  To  the  remainder  of  the  solution  add  a  little  dilute 
hydrochloric  acid  and  boil  for  a  few  moments.  Cool  the  solu- 
tion, neutralize  with  solid  KOH  and  try  Fehling's  test.  Ex- 
plain the  result. 

IV.     OSSEOUS    TISSUE. 

Bone  is  composed  of  about  equal  parts  of  organic  and  in- 
organic matter.  The  organic  portion,  called  ossein,  may  be 
obtained  by  removing  the  inorganic  salts  through  the  medium 
of  dilute  acid.  Ossein  is  practically  the  same  body  which  is 
termed  collagen  in  the  other  connective  tissues,  and  in  com- 
mon with  collagen  may  be  hydrolyzed  with  weak  acids  to  form 
gelatin. 

In  common  with  the  other  connective  tissues  bone  contains 
a  mucoid  and  an  albumoid.  Because  of  their  origin  these 
bodies  are  called  osseomucoid  and  osseoalbumoid.  Osseo- 
mucoid, when  boiled  with  hydrochloric  acid,  yields  sulphuric 
acid  and  a  substance  capable  of  reducing  Fehling's  solution. 
The  composition  of  osseomucoid  is  very  similar  to  that  of 
tendomucoid  and  chondromucoid  (see  page  62). 

Experiment  on  Osseous  Tissue. 

Analysis  of  Bone  Ash. — Take  one  gram  of  bone  ash  in 
a  small  beaker  and  add  a  little  dilute  nitric  acid.  What  does 
the  effervescence  indicate?  Stir  thoroughly  and  when  the 
major  portion  of  the  ash  is  dissolved  add  an  equal  volume  of 
water  and  filter.  To  the  acid  filtrate  add  ammonium  hydroxide 
to  alkaline  reaction.  A  heavy  white  precipitate  of  phosphates 
results.  (What  phosphates  are  precipitated  here  by  the 
ammonia?)  Filter  and  test  the  filtrate  for  chlorides,  sul- 
phates, phosphates  and  calcium.  Add  dilute  acetic  acid  to  the 
precipitate  on  the  paper  and  test  this  filtrate  for  calcium  and 
phosphates.  To  the  precipitate  remaining  undissolved  on  the 
paper  add  a  little  dilute  hydrochloric  acid  and  test  this  last 
filtrate  for  phosphates  and  iron. 

Reference  to  the  following  scheme  may  facilitate  the  analysis. 


EPITHELIAL    AND    CONNECTIVE    TISS1   ES.  205 

BONE   ASH. 

Add  dilute  IINO3,  stir  thoroughly  and  after  the  major  portion  of  the 
ash  lias  been  brought  into  solution  add  a  little  distilled  water  and  filter. 


Residue   I.  Filtrate  I. 

(discard)  Add  NH4OH  to  alka- 

line reaction  and  filter. 


Residue  II. 
Treat  on  paper  with  acetic  acid. 


Residue  III.  Filtrate  III. 

Treat  on  paper  with       Test  for: 
HC1.  1.  Phosphates. 

2.  Calcium. 

Filtrate  IV. 
Test  for: 

1.  Iron. 

2.  Phosphates. 


Filtrate  II. 
Test  for: 

1.  Chlorides. 

2.  Sulphates. 

3.  Phosphates. 

4.  Calcium. 


V.     ADIPOSE   TISSUE. 

For  discussion  and  experiments  see  the  chapter  on  Fats, 
page  96. 


CHAPTER   XIV. 
MUSCULAR  TISSUE. 

The  muscular  tissues  are  divided  physiologically  into  the 
voluntary  and  the  involuntary.  In  the  chemical  examination 
of  muscular  tissue  the  voluntary  form  is  generally  employed. 
Muscle  contains  about  25  per  cent  of  solid  matter,  of  which 
about  four-fifths  is  proteid  material  and  the  remaining  one- 
fifth  extractives  and  inorganic  salts. 

The  proteids  are  the  most  important  of  the  constituents  of 
muscular  tissue.  In  the  living  muscle  we  find  two  proteids, 
myosinogen  and  para-myosinogen.  These  may  be  shown  to 
be  present  in  muscle  plasma  expressed  from  fresh  muscles. 
In  common  with  the  plasma  of  the  blood  this  muscle  plasma 
has  the  power  of  coagulating,  and  the  clot  formed  in  this 
process  is  called  myosin.  In  the  onset  of  rigor  mortis  we  have 
an  indication  of  the  formation  of  this  myosin  clot  within  the 
body.  The  relation  between  the  proteids  of  living  and  dead 
muscle  is  represented  graphically  by  Halliburton  as  follows : 

Proteids  of  the  living  muscle. 


Para-myosinogen.  Myosinogen. 

Soluble  myosin. 


\/ 

Myosin. 

(The  proteid  of  the  muscle  clot.) 

Of  the  total  proteid  content  of  living  muscle  about  75  per 
cent  is  made  up  by  the  myosinogen  and  the  remaining  25  per 
cent  is  para-myosinogen.  These  proteids  may  be  separated 
by  subjecting  the  muscle  plasma  to  fractional  coagulation  in 
the  usual  way.     Under  these  conditions  the  para-myosinogen 

206 


M  USCULAR    TISS1   E.  -"7 

is  found  to  coagulate  at  47  C.  and  the  myosinogen  to  coagu- 
late at  560  C.  It  is  also  claimed  by  some  investigators  that 
it  is  possible  to  separate  these  two  proteids  by  the  fractional 
ammonium  sulphate  method,  but  the  possibility  of  making  an 
accurate  separation  by  this  method  is  somewhat  doubtful.  It 
is  well  established  that  para-myosinogen  is  a  globulin  since  it 
responds  to  .certain  of  the  proteid  precipitation  tests  and  is 
insoluble  in  water.  Myosinogen,  on  the  contrary,  is  not  a 
typical  globulin  since  it  is  soluble  in  water.  It  has  been  called 
a  pseudo-globulin.  Myosin  possesses  the  globulin  character- 
istics. It  is  insoluble  in  water  but  soluble  in  the  other  proteid 
solvents  and  is  precipitated  from  its  solution  upon  saturation 
with  sodium  chloride. 

Under  the  name  extractives  we  class  a  number  of  muscle 
constituents  which  occur  in  traces  in  the  tissue  and  may  be 
extracted  by  water,  alcohol  or  ether.  There  are  two  classes 
of  these  extractives,  the  non-nitrogenous  extractives  and  the 
nitrogenous  extractives.  Grouped  under  the  non-nitrogenous 
bodies  we  have  glycogen,  dextrin,  sugars,  lactic  acid,  inosit, 
C6Hc(OH)6,  and  fat.  In  the  class  of  nitrogenous  extractives 
we  have  crcatin,  creatinin,  xanthin,  hypoxanthin,  uric  acid, 
urea,  carnin,  phosphocarnic  acid,  inosinic  acid,  carnosin  and 
taurin  (see  formulas  on  page  210).  Not  all  of  these  extrac- 
tives are  present  in  the  muscles  of  all  species  of  animals.  Other 
extractives  besides  those  enumerated  above  have  been  described 
and  there  are  undoubtedly  still  others  whose  presence  remains 
undetermined.  A  detailed  consideration  wrould  however  be 
unprofitable  in  this  place. 

Glycogen  is  an  important  constituent  of  muscle.  The  con- 
tent of  this  polysaccharide  in  muscle  varies  and  is  markedly 
decreased  by  intense  muscular  activity.  It  is  transformed  into 
sugar  and  used  as  fuel.  The  liver  is  the  organ  which  stores 
the  reserve  supply  of  glycogen  and  transforms  it  into  dextrose 
which  is  passed  into  the  blood  stream  and  so  carried  to  the 
working  muscle  where  it  is  synthesized  into  glycogen.  The 
glycogen  thus  formed  is  then  changed  into  dextrose  as  the 
working  muscle  may  need  it. 


208  PHYSIOLOGICAL    CHEMISTRY. 

Glycogen  is  a  polysaccharide  and  has  the  same  percentage 
composition  as  starch  and  dextrin.  It  resembles  starch  in 
forming  an  opalescent  solution  and  resembles  dextrin  in  being 
very  soluble,  in  giving  a  reddish  color  with  iodine  and  in  being 
dextro-rotatory.  Glycogen  may  be  prepared  from  muscle  by 
extracting  with  boiling  water  and  then  precipitating  the  gly- 
cogen from  the  aqueous  solution  by  alcohol :  dilute  or  concen- 
trated KOH  may  also  be  used  to  extract  the  glycogen.  Gly- 
cogen may  be  prepared  in  the  form  of  a  white,  tasteless,  amor- 
phous powder.  It  is  completely  precipitated  from  its  solution  by 
saturation  with  solid  ammonium  sulphate,  but  is  notprecipitated 
by  saturation  with  sodium  chloride.  It  may  also  be  precipitated 
by  alcohol,  tannic  acid  or  ammoniacal  basic  lead  acetate.  It  has 
the  power  of  holding  cupric  hydroxide  in  solution  in  alkaline 
fluids  but  cannot  reduce  it.  It  may  be  hydrolyzed  with  the 
formation  of  dextrose  by  dilute  mineral  acids  and  is  readily 
digested  by  amylolytic  enzymes. 

The  lactic  acid  occurring  in  the  muscular  tissue  of  verte- 
brates is  paralactic  or  sarcolactic  acid, 

H     OH 
H  — C  — C  — COOH. 
H     H 

The  reaction  of  an  inactive  living  muscle  is  alkaline,  but  upon 
the  death  of  the  muscle,  or  after  the  continued  activity  of  a 
living  muscle,  the  reaction  becomes  acid,  due  to  the  formation 
of  lactic  acid.  There  is  a  difference  of  opinion  regarding  the 
origin  of  this  lactic  acid.  Some  investigators  claim  it  to  arise 
from  the  carbohydrates  of  the  muscle,  while  others  ascribe  to 
it  a  proteid  origin. 

Among  the  nitrogenous  extractives  of  muscle,  those  which 
are  of  the  most  interest  in  this  connection  are  creatin  and  the 
purin  bases,  xanthin  and  hypoxanthin.  Creatin  is  found  in 
varying  amounts  in  the  muscles  of  different  species,  the  mus- 


MUSCII.AK    TISSUE. 


209 


cles  of  birds  having  shown  the  largest  amount.  It  has  also 
been  found  in  the  blood,  the  brain,  in  transudates  and  in  the 
thyroid  gland.  Creatin  may  be  crystallized  and  forms  color- 
less  rhombic  prisms  (Fig.  yj,  below)  which  are  soluble  in 
warm  water  and  practically  insoluble   in  alcohol  and  ether. 

Fig.  77- 


Creatin. 


Upon  boiling  a  solution  of  creatin  with  dilute  hydrochloric 
acid  it  is  dehydrolyzed  and  its  anhydride  creatinin  is  formed. 
The  creatin  of  ingested  meat  is  transformed  into  creatinin 
and  excreted  in  the  urine. 

Besides  being  a  normal  constituent  of  muscle,  xanthin  has 
been  found  in  the  brain,  spleen,  pancreas,  thymus,  kidneys, 
testicles,  liver,  and  in  the  urine.  It  may  be  obtained  in  crys- 
talline form  (Fig.  78,  p.  210)  but  ordinarily  it  is  amorphous. 
Xanthin  is  easily  soluble  in  alkalis,  less  easily  soluble  in  water 
and  dilute  acids,  and  entirely  insoluble  in  alcohol  and  ether. 

Hypoxanthin  occurs  ordinarily  in  those  tissues  and  fluids 

which  contain  xanthin.     It  has  been  found,  unaccompanied  by 

xanthin,  in  bone  marrow  and  in  milk.     Unlike  xanthin  it  may 

be  easily  crystallized  in  the  form  of  small,  colorless  needles. 

'5 


2IO 


PHYSIOLOGICAL    CHEMISTRY. 


I:  is  readily  soluble  in  alkalis,  acids  and  boiling  water,  less 
soluble  in  cold  water  and  practically  insoluble  in  alcohol  and 
ether. 

The  predominating  inorganic  salt  of  muscle  is  potassium 
phosphate.  Besides  this  salt  we  have  present  sulphates,  chlo- 
rides and  salts  of  sodium,  calcium,  magnesium  and  iron. 

Fig.  78. 


Xanthin. 
After  the  drawings  of  Horbaczewski,  as  represented  in  Xeubauer  and  Vogel. 

(Ogden.) 

Muscular  tissue  is  said  to  contain  a  reddish  pigment  called 
my  ohcc  matin,  which  is  a  derivative  of.. haemoglobin. 

The  ordinary  commercial  "  meat  extract  "  is  composed  prin- 
cipally of  the  water-soluble  constituents  of  muscle  and  con- 
tains practically  nothing  of  nutritive  value.  The  proteid  mate- 
rial to  which  meat  owes  its  value  as  an  article  of  diet  is 
practically  all  removed  in  the  preparation  of  the  extract. 

The  structural  formulas  of  the  nitrogenous  extractives  of 
muscle  are  as  follows : 


NH, 


HN- 


■00 


HN  =  C 


HN  — C 


N-CBU-CEL-COOH 


Creatin,  C4Ht,N302. 
Methyl-guanidin  acetic  acid. 


N-CH8-CHS 

Creatinin,  C|H7N30. 
Creatin  anhydride. 


XH., 

i 

1 

0 

XHo 

Urea, 

CON2H4. 

M  rsciT.AU    TISSIK.  2  1  I 


cil-xii 


CH,  •  S02  •  OH 

TaURIN,    C2II7NSO3. 

A  mino-cthylsuJ  phonic  acid. 


Camosin,  C9H14X403. 

Inosinic  acid,  C]0H13XT4P08. 

Phosphocarnic  acid,  Clniri7X305  °r  C,nII,-X 8( ">.-.. 

The  following  extractives  as  a  group  are  called  purin 
bodies.  Their  formulas,  together  with  that  of  purin  from 
which  they  are  derived  and  the  hypothetical  "  purin  nucleus  " 
follow  : 

X  =  CH  *X  —  C° 

II  II 

HC      C  — XH  2C        C5-X7 


CH 


\n 


II         II            /^-n-  I            I               /-s 

N  — C  — N  3X—  C4  —  Nn 

Purin,  C0H4N4.  Purin  Nucleus. 

HX  — CO  HX  — CO 

II  II 

HC      C  — XH  OC      C  — XH 

II       II         )CH  I        ||         )CH 

N  — C  — X  HX  — C  — X 

Hypoxantrin,  C«H4N40.  Xanthin,  C5H4N4O2. 

6-oxypurin.  Uoxypurin. 

HX  — CO  X  —  C-NHo 

II  II" 

OC      C  — XH  HC      C  — XH 

I       II         >C0  II       II         )CH 

HX  — C  — XH  X  —  C  —  X 

Uric  Acid,  Q.IL.NiO.-,.  Adenin,  CbH5Nb. 

3-6-S-trioxy  purin.  6-aminopurin. 


212  PHYSIOLOGICAL    CHEMISTRY. 

HN  — CO 

I        I 
HoN-C      C  — NH 

II       II  )CH 

N  — C  — N 

Guanin,   C5H5N50. 
z-amino-6-oxypurin. 

Experiments  on  Muscular  Tissue. 

I.  Experiments  on  "Living"  Muscle. 

I.  Preparation  of  Muscle  Plasma  (Halliburton). — Wash 
out  the  blood  vessels  of  a  freshly  killed  rabbit  with  0.9  per 
cent  sodium  chloride.  This  can  best  be  done  by  opening  the 
abdomen  and  inserting  a  cannula  into  the  aorta.  Now  re- 
move the  skin  from  the  lower  limbs,  cut  away  the  muscles  and 
divide  them  into  very  small  pieces  by  means  of  a  meat 
chopper.  Transfer  the  pieces  of  muscle  to  a  mortar  and  grind 
them  with  clean  sand  and  a  little  5  per  cent  magnesium  sul- 
phate. Filter  off  the  salted  muscle  plasma  and  make  the  fol- 
lowing tests : 

(a)  Reaction. — Test  the  reaction  to  litmus.  What  is  the 
reaction  of  this  fresh  muscle  plasma? 

(b)  Fractional  Coagulation. — Place  a  little  muscle  plasma 
in  a  test-tube  and  arrange  the  apparatus, .for  fractional  coagu- 
lation as  explained  on  page  50.  Raise  the  temperature  very 
carefully  from  300  C.  and  note  any  changes  which  may  occur 
and  the  exact  temperature  at  which  such  changes  take  place. 
When  the  first  proteid  (para-myosinogen)  coagulates  filter  it 
off  and  then  heat  the  clear  filtrate  as  before,  being  careful  to 
note  the  exact  temperature  at  which  the  next  coagulation 
(myosinogen)  occurs.  There  will  probably  be  a  preliminary 
opalescence  in  each  case  before  the  real  coagulation  occurs. 
Therefore  do  not  mistake  the  real  coagulation-point  and  filter 
at  the  wrong  time.  What  are  the  coagulation  temperatures 
of  these  two  proteids?  Which  proteid  was  present  in  greater 
amount  ? 


MUSCULAR    TISSUE.  213 

(c)  Formation  of  the  Myosin  Clot. — Dilute  a  portion  of 
the  plasma  with  3  or  4  times  its  volume  of  water  and  place 
it  on  a  water-bath  or  in  an  incubator  at  35°  C.  for  several 
hours.  A  typical  myosin  clot  should  form.  Note  the  muscle 
serum  surrounding  the  clot.  Now  test  the  reaction.  Has  the 
reaction  changed,  and  if  so, to  what  is  the  change  due?  Make 
a  test  for  lactic  acid.     What  do  you  conclude? 

2.  Preparation  of  Muscle  Plasma  (v.  Fiirth). —  Remove 
the  blood-free  muscles  of  a  rabbit  as  explained  on  page  212. 
Finely  divide  by  means  of  a  meat  chopper  and  grind  in  a 
mortar  with  a  little  clean  sand  and  some  0.9  per  cent  sodium 
chloride.  Wrap  portions  of  the  muscle  in  muslin  and  press 
thoroughly  by  means  of  a  tincture  press  or  lemon  squeezer. 
Filter  and  make  the  tests  according  to  the  directions  given  in 
the  last  experiment. 

3.  "  Fuchsin-Frog  "  Experiment. — Inject  a  saturated 
aqueous  solution  of  Fuchsin  "S"  into  the  lymph  spaces  of  a  frog 
three  or  four  times  daily  for  two  or  three  days,  in  this  way 
thoroughly  saturating  the  tissues  with  the  dye.  Pith  the  animal 
(insert  a  heavy  wire  or  blunt  needle  through  the  occipito  atlan- 
toid  membrane),  remove  the  skin  from  both  hind  legs  and 
expose  the  sciatic  nerve  in  one  of  them.  Insert  a  small  wire 
hook  through  the  jaws  of  the  frog  and  suspend  the  animal 
from  an  ordinary  clamp  or  iron  ring.  Pass  electrodes  under 
the  exposed  sciatic  nerve,  and  after  tying  the  other  leg  to  pre- 
vent any  muscular  movement,  stimulate  the  exposed  nerve  by 
means  of  make  and  break  shocks  from  an  induction  coil.  The 
stimulated  leg  responds  by  pronounced  muscular  contractions, 
whereas  the  tied  leg  remains  inactive.  Continue  the  stimula- 
tion until  the  muscles  are  fatigued.  The  muscular  activity 
has  caused  the  production  of  lactic  acid  and  this  in  turn 
has  reacted  with  the  injected  fuchsin  to  cause  a  pink  or  red 
color  to  develop.  The  muscles  of  the  inactive  leg  still  remain 
unchanged  in  color. 

The  normal  color  of  the  Fuchsin  "  S  "  when  injected  was 
red,  but  upon  being  absorbed  it  became  colorless  through  the 


214  PHYSIOLOGICAL    CHEMISTRY. 

action  of  the  alkalinity  of  the  blood.  Upon  stimulating  the 
muscles,  however,  as  above  explained,  lactic  acid  was  formed 
and  this  acid  reacted  with  the  fuchsin  and  again  produced  the 
original  color  of  the  dye. 

II.  Experiments  on  "  Dead  "  Muscle. 

i.  Preparation  of  Myosin. — Take  25  grams  of  finely 
divided  lean  beef  which  has  been  carefully  washed  to  remove 
blood  and  lymph  constituents  and  place  it  in  a  beaker  with  10 
per  cent  sodium  chloride.  Stir  occasionally  for  several  hours. 
Strain  off  the  meat  pieces  b}^  means  of  cheese  cloth,  filter  the 
solution  and  saturate  it  with  sodium  chloride  in  substance. 
Filter  off  the  precipitate  of  myosin  and  make  the  tests  as  given 
below.  This  filtration  will  proceed  very  slowly.  Myosin 
collects  as  a  film  on  the  sides  of  the  filter  paper  and  may  be 
removed  and  tested  before  the  entire  volume  of  fluid  has  been 
filtered.     Test  the  myosin  precipitate  as  follows : 

(a)  Solubility. — Try  its  solubility  in  the  ordinary  solvents. 
Is  myosin  an  albumin  or  a  globulin? 

(b)  Xanthoproteic  Reaction. — See  page  44. 

(c)  Coagulation  Test. — Suspend  a  little  of  the  myosin  in 
water  in  a  test-tube  and  heat  to  boiling  for  a  few  moments. 
Now  remove  the  suspended  material  and  try  its  solubility  in 
10  per  cent  sodium  chloride.  What  property  does  this  ex- 
periment show  myosin  to  possess  ? 

Test  the  filtrate  from  the  original  myosin  precipitate  as 
follows : 

(a)  Biuret  Test. — What  does  this  show? 

(b)  Place  a  little  of  the  solution  in  a  test-tube  and  heat 
to  boiling.  At  the  boiling-point  add  a  drop  of  dilute  acetic 
acid  and  filter.  Test  this  filtrate  for  proteose  with  picric 
acid.  Is  any  proteose  present?  Saturate  another  portion  of 
the  filtrate  with  ammonium  sulphate  and  test  for  peptone  in 
the  usual  way  (see  page  59).  Do  you  find  any  peptone? 
From  your  experiments  on  "  living  "  and  "  dead  "  muscle  what 
are  your  ideas  regarding  the  proteids  of  muscle? 


MUSCULAR    TISSUE.  215 

2.  Preparation  of  Glycogen. — Grind  a  few  scallops  in  a 
mortar  with  sand.  Transfer  to  an  evaporating  dish,  add  water 
and  boil  for  20  minutes.  At  the  boiling-point  faintly  acidify 
with  acetic  acid.  Why  is  this  acid  added?  Filter,  and  divide 
the  nitrate  into  two  parts.  Note  the  opalescence  of  the  solu- 
tion.   Test  one  portion  of  the  filtrate  as  follows : 

( (/ )  Iodine  Test. — To  5  c.c.  of  the  solution  in  a  test-tube 
add  2-3  drops  of  iodine  solution  and  2-3  drops  of  10  per  cent 
sodium  chloride.  Warm  this  slightly  and  then  allow  it  to 
cool.  What  do  you  observe?  Is  this  similar  to  the  iodine  test 
upon  any  other  body  with  which  we  have  had  to  deal? 

(b)  Reduction  Test. — Does  the  solution  reduce  Fehling's 
solution? 

(c)  Hydrolysis  of  Glycogen. — Add  10  drops  of  concen- 
trated hydrochloric  acid  to  10  c.c.  of  the  solution  and  boil  for 
10  minutes.  Cool  the  solution,  neutralize  with  solid  potassium 
hydroxide  and  test  with  Fehling's  solution.  Does  it  still  fail 
to  reduce  Fehling's  solution?  If  you  find  a  reduction  how  can 
you  prove  the  identity  of  the  reducing  substance? 

(d)  Influence  of  Salizv. — Place  5  c.c.  of  the  solution  in  a 
test-tube,  add  5  drops  of  saliva  and  place  on  the  water-bath  at 
400  C.  for  10  minutes.  Does  this  now  reduce  Fehling's 
solution? 

To  the  second  part  of  the  glycogen  filtrate  add  3-4  volumes 
of  95  per  cent  alcohol.  Allow  the  glycogen  precipitate  to 
settle,  decant  the  supernatant  fluid,  filter  the  remainder  and 
upon  the  glycogen  make  the  following  tests : 

(a)  Solubility. — Try  its  solubility  in  the  ordinary  solvents. 

(b)  Iodine  Test. — Place  a  small  amount  of  the  glycogen  in 
a  depression  of  a  test-tablet  and  add  a  drop  of  dilute  iodine 
solution  and  a  trace  of  a  sodium  chloride  solution.  The  same 
wine-red  color  is  observed  as  in  the  iodine  test  upon  the 
glycogen  solution. 

Separation  of  Extractives  from  Muscle. 
1.  Creatin. — Dissolve  about  10  gram-  of  a  commercial  ex- 
tract of  meat  in  200  c.c.  of  warm  water.     Precipitate  the  inor- 


2l6 


PHYSIOLOGICAL    CHEMISTRY. 


ganic  constituents  by  neutral  lead  acetate,  being  careful  not  to 
add  an  excess  of  the  reagent.  Write  the  equations  for  the 
reactions  taking  place  here.  Allow  the  precipitate  to  settle., 
then  filter  and  remove  the  excess  of  lead  in  the  warm  filtrate 
by  H2S.  Filter  while  the  solution  is  yet  warm,  evaporate  the 
clear  filtrate  to  a  syrup  and  allow  it  to  stand  at  least  48  hours 
in  a  cool  place.  Crystals  of  creatin  should  form  at  this  point. 
Examine  under  the  microscope  (Fig.  yy,  page  209).  Treat 
the  syrup  with  200  c.c.  of  88  per  cent. alcohol,  stir  well  with  a 
glass  rod  to  bring  all  soluble  material  into  solution,  and  then 
filter.  The  purin  bases  have  been  dissolved  and  are  in  the 
filtrate,  whereas  the  creatin  crystals  were  insoluble  in  the  88 
per  cent  alcohol  and  remain  on  the  filter  paper.  Wash  the 
crystals  with  88  per  cent  alcohol,  then  remove  them  and  bring 

Fig.  79. 


Hypoxaxtiiix  Silver  Nitrate. 


them  into  solution  in  a  little  hot  water.  Decolorize  the  solu- 
tion by  animal  charcoal  and  concentrate  it  to  a  small  volume. 
Allow  the  solution  to  cool  and  note  the  separation  of  colorless 
crystals  of  creatin.  Examine  these  crystals  under  the  micro- 
scope and  compare  them  with  those  reproduced  in  Fig.  yy, 
page  209. 


MUSCULAR    TISSUE.  -17 

2.  Hypoxanthin. — Evaporate  the  alcoholic  filtrate  from 
the  creatin  to  remove  the  alcohol.  Make  the  solution  annnn- 
niacal  and  add  ammoniacal  silver  nitrate  until  precipitation 
ceases.  The  precipitate  consists  principally  of  hypoxanthin 
silver  and  xanthin  silver.  Collect  these  silver  salts  on  a  filter 
paper  and  wash  them  with  water.  Place  the  precipitate  and 
paper  in  an  evaporating  dish  and  boil  for  one  minute  with 
nitric  acid  having  a  specific  gravity  of  t.t.  Filter  while  hot 
through  a  double  paper,  wash  with  the  same  strength  of 
nitric  acid  and  allow  the  solution  to  cool.  By  this  treat- 
ment with  nitric  acid  hypoxanthin  silver  nitrate  and  xan- 
thin silver  nitrate  have  been  formed.  The  former  is  in- 
soluble in  the  cold  solution  and  separates  on  standing.  After 
standing  several  hours  filter  off  the  hypoxanthin  silver  nitrate 
and  wash  with  water  until  the  wash-water  is  only  slightly  acid 
in  reaction.  Examine  the  crystals  of  hypoxanthin  silver  ni 
trate  under  the  microscope  and  compare  them  with  those  in 
Fig.  79.  page  216.  Now  wash  the  crystals  from  the  paper  into 
a  beaker  with  a  little  water  and  warm  the  liquid.  Remove  the 
silver  by  H2S  and  filter.  By  this  means  hypoxanthin  nitrate 
has  been  formed  and  is  present  in  the  filtrate.  Concentrate  on 
a  water-bath  to  drive  off  hydrogen  sulphide  and  render  the 
solution  slightly  alkaline  with  ammonia.  Warm  for  a  time, 
to  remove  the  free  ammonia,  filter,  concentrate  the  filtrate  to  a 
small  volume  and  allowr  it  to  stand  in  a  cool  place.  Hypox- 
anthin should  crystallize  in  small  colorless  needles.  Examine 
the  crystals  under  the  microscope. 

3.  Xanthin. — To  the  filtrate  from  the  above  experiment 
containing  the  xanthin  silver  nitrate  add  ammonia  in  excess. 
(The  crystalline  form  of  xanthin  silver  nitrate  is  shown  in 
Fig.  80,  p.  218.)  A  brownish-red  precipitate  of  xanthin  silver 
forms.  Treat  this  suspended  precipitate  with  H2S  (do  not 
use  an  excess  of  H2S),  warm  the  mixture  for  a  few  moments 
and  filter  while  hot.  Concentrate  the  filtrate  to  a  small  volume 
and  put  away  in  a  cool  place  for  crystallization  (Fig.  78,  p.  210). 
To  obtain  xanthin  in  crystalline  form  special  precautions  are 


2l8  PHYSIOLOGICAL    CHEMISTRY. 

generally    necessary.     Evaporate    the    solution    to    dryness. 
Make  the  following  tests  on  the  crystals  or  residue : 

(o)  Xanthin  Test. — Place  about  one-half  of  the  crystalline 
or  amorphous  material  in  a  small  evaporating  dish,  add  a  few 

Fig.  80. 


Xanthin  Silver  Nitrate. 

drops  of  concentrated  nitric  acid  and  evaporate  to  dryness 
very  carefully  on  a  water-bath.  The  yellow  residue  upon 
moistening  with  caustic  potash  becomes  red  in  color  and  upon 
further  heating  assumes  a  purplish-red  hue.  Now  add  a  few 
drops  of  water  and  warm.  In  this  way  a  yellow  solution  re- 
sults which  yields  a  red  residue  upon  evaporation.  How  does 
this  differ  from  the  Murexid  test  upon  uric  acid  ? 

(b)  WeideVs  Reaction. — By  gently  heating  bring  the  re- 
mainder of  the  xanthin  crystals  or  residue  into  solution  in 
bromine-water.  Evaporate  the  solution  to  dryness  on  a  water- 
bath.  Remove  the  stopper  from  an  ammonia  bottle  and  by 
blowing  across  the  mouth  of  the  bottle  direct  the  fumes  of 
ammonia  so  that  they  come  in  contact  with  the  dry  residue. 
Under  these  conditions  the  presence  of  xanthin  is  shown  by 
the  residue  assuming  a  red  color.  A  somewhat  brighter  color 
may  be  obtained  by  using  a  trace  of   nitric   acid  with  the 


MI'Mi   i.  \K    TISSUE.  219 

bromine-water.  By  the  use  of  this  modification  however  we 
may  get  a  positive  reaction  with  bodies  other  than  xanthin. 

Hurtiile's  Ex  pi-rim  ent. 
Tease  a  very  small  piece  of  frog's  muscle  on  a  microscopical 
slide.  Expose  the  slide  to  ammonia  vapor  for  a  few  moments, 
then  adjust  a  cover  glass  and  examine  the  muscle  fibers  under 
the  microscope.  Note  the  large  number  of  crystals  of  ammo- 
nium magnesium  phosphate, 

NH4  — 0 

\ 
Mg  —  0  —  P  =  0 
\      / 
O 

distributed  everywhere  throughout  the  muscle  fiber,  thus 
demonstrating  the  abundance  of  phosphates  and  magnesium 
in  the  muscle  (Fig.  96,  page  278). 


CHAPTER   XV. 
NERVOUS  TISSUE. 

In  common  with  the  other  solid  tissues  of  the  body,  nervous 
tissue  contains  a  large  amount  of  water.  The  percentage  of 
water  present  depends  upon  the  particular  form  of  nervous 
tissue  but  in  all  forms  it  is  invariably  greater  in  the  gray  matter 
than  in  the  white.  Embryonic  nervous  tissues  also  contain  a 
larger  percentage  of  water  than  the  tissues  of  adult  life.  The 
gray  matter  of.  the  brain  of  the  foetus,  for  instance,  contains 
about  92  per  cent  of  water,  whereas  the  gray  matter  of  the 
brain  of  the  adult  contains  but  83-84  per  cent  of  the  fluid. 

Among  the  solid  constituents  of  nervous  tissue  are  proteids, 
cholesterin,  cerebrin,  lecithin,  kephalin,  protagon{?) ,  nuclein, 
neuro-keratin,  collagen/  extractives  and  inorganic  salts.  The 
proteids  are  present  in  the  greatest  amount  and  comprise  about 
50  per  cent  of  the  total  solids.  Three  distinct  proteids,  two 
globulins  and  a  nucleo-proteid,  have  been  isolated  from  ner- 
vous tissue.  The  globulins  coagulate  at  47  °  C.  and  70-75  °  C. 
respectively,  while  the  nucleo-proteid  coagulates  at  56-600  C. 
This  nucleo-proteid  contains  about  0.5  per  cent  of  phosphorus 
(Halliburton,  Levene).  Nervous  tissue  is  composed  of  a  rela- 
tively large  quantity  of  a  variety  of  compounds  which  col- 
lectively may  be  grouped  under  the  term  "lipoid" — substances 
resembling  the  fats  in  some  of  their  physical  properties  and 
reactions  but  distinct  in  their  composition.  We  will  class 
cerebrin,  cholesterin  and  the  phosphorized  fats,  as  "lipoids." 

The  group  of  phosphorized  fats  are  very  important  con- 
stituents of  nervous  tissue.  The  best  known  members  of  this 
group  arc  lecithin,  protagon  ( ?)  and  kephalin.  Lecithin  occurs 
in  larger  amount  than  the  other  members  of  the  group,  has 
been  more  thoroughly  studied  than  the  others  and  is  apparently 
of  greater  importance.     Upon  decomposition  lecithin  yields 


N  ERVOUS     l  ISSUE.  22  1 

fatty  acid,  glycero-phosphoric  acid  and  cholin.    Each  lecithin 

molecule  contains  two  Tatty  acid  radicals  which  may  he  those 
of  the  same  or  different  fatty  acids.  Thus  we  have  different 
lecithins  depending  upon  the  particular  fatty  acid  radicals 
which  are  present  in  the  molecule.  The  formula  of  a  typical 
lecithin  would  be  the  following: 

CHoO  — C17H,5CO 

CHO  —  C17H35CO 

CHoO  — PO  — 0-C2H4 

(CH3)iN 
OH  HO  ' 

This  lecithin  would  be  called  distearyl-lecithin  or  cholin-dis- 
tearyl-glycero-phosphoric  acid.  Upon  decomposition  the  mole- 
cule splits  according  to  the  following  reaction : 

C44H90NPO0  +,3H20=2(C18H3602)  + 

Lecithin.  Stearic  acid. 

C3H9P06  +  C5H]5X02. 

Glycero-phosphoric  Cholin. 

acid. 

The  lecithins  are  not  confined  to  the  nervous  tissues  but 
are  found  in  nearly  all  animal  and  vegetable  tissues.  Lecithin 
is  a  primary  constituent  of  the  cell.  It  is  soluble  in  chloro- 
form, ether,  alcohol,  benzene  and  carbon  disulphide.  The 
chloroform  or  alcohol-ether  solution  may  be  precipitated  by 
acetone.  Lecithin  may  be  caused  to  crystallize  in  the  form 
of  small  plates  by  cooling  the  alcoholic  solution  to  a  low  tem- 
peratures It  has  the  power  of  combining  with  acids  and  bases, 
and  the  hydrochloric  acid  combination  has  the  power  of  form- 
ing a  double  salt  with  platinic  chloride. 

Protagon,  another  nitrogenous  phosphorized  substance  is  a 
body  over  which  there  has  been  much  discussion.  L'pon  de- 
composition it  is  said  by  some  investigators  to  yield  cerebrin 


222  PHYSIOLOGICAL    CHEMISTRY. 

and  the  decomposition  products  of  lecithin.  It  has  very 
recently  been  shown  by  Posner  and  Gies  that  protagon  is  a 
mixture  and  has  no  existence  as  a  chemical  individual. 

Kephalin  is  the  third  member  of  the  group  of  phosphorized 
fats.  It  is  precipitated  from  its  acetone-ether  extract  by 
alcohol.  It  contains  about  4  per  cent  of  phosphorus  and  has 
been  given  the  formula  C42H79NP013.  Kephalin  may  be  a 
stage  in  lecithin  metabolism. 

Cerebrin,  a  substance  containing  nitrogen  but  no  phos- 
phorus, is  an  important  constituent  of  the  white  matter  of 
nervous  tissue.  It  has  also  been  found  in  the  spleen,  pus 
and  in  egg  yolk.  It  may  be  extracted  from  the  tissue  by  boil- 
ing alcohol  and  is  insoluble  in  cold  alcohol,  cold  and  hot  ether 
and  in  water  and  dilute  alkalis.  Cerebrin  is  a  mixture  con- 
taining phrenosin  (pseudo-cerebrin  or  cerebron),  a  body  yield- 
ing the  carbohydrate  galactose  on  decomposition. 

Cholesterin,  one  of  the  primary  cell  constituents,  is  present 
in  fairly  large  amount  in  nervous  tissue.  It  is  a  mon-atomic 
alcohol  with  the  formula  C27H45OH.  It  was  formerly  called 
a  "  non-saponifiable  fat "  but  since  it  is  not  changed  in  any 
way  by  boiling  alkalis  it  is  not  a  fat.  It  is  soluble  in  ether, 
chloroform,  benzene  and  hot  alcohol.  It  crystallizes  in  the 
form  of  thin,  colorless,  transparent  plates  (Fig.  42,  p.  — ). 
Cholesterin  occurs  abundantly  in  one  form  of  biliary  calculus. 
It  has  also  been  found  in  feces,  wool  iat,  egg  yolk,  and  milk, 
frequently  in  the  form  of  its  esters  of  higher  fatty  acids. 

Nervous  tissue  yields  about  1  per  cent  of  ash  which  is  made 
up  in  great  part  of  alkaline  phosphates  and  chlorides. 

Experiments  on  the  Lipoids  of  Nervous  Tissue.1 

1.  Preparation  of  Lecithin. — Treat  the  macerated  brain 

of  a  sheep  with  ether  and  allow  it  to  stand  in  the  cold  for 

1  Preparation  of  So-called  Protagon. — Macerate  the  brain  of  a  sheep,  treat 
with  85  per  cent  alcohol  and  warm  on  a  water-bath  at  45°  C.  for  two  hours. 
Filter  hot  into  a  bottle  or  strong  flask  and  cool  to  0°  C.  for  one-half  hour  by 
means  of  a  freezing  mixture.  By  this  procedure  both  protagon  and  choles- 
terin are  caused  to  precipitate.  Filter  the  cold  solution  rapidly  and  treat 
the  precipitate  on  the  paper  with  ice  cold  ether  to  dissolve  out  the  choles- 
terin. The  protagon  may  now  be  redissolved  in  warm  85  per  cent  alcohol 
from  which  solution  it  will  precipitate  upon  cooling. 


NERVOUS   tissue.  223 

48-72  hours.  The  cold  ether  will  extract  lecithin  and  choles- 
terin.  Filter,  and  t\(](\  acetone  to  the  filtrate  to  precipitate  the 
lecithin.     Filter  off  the  lecithin  and  test  it  as  follows: 

(a)  Microscopical  Examination. — Suspend  a  small  portion 
in  a  drop  of  water  on  a  slide  and  examine  under  the  micro- 
scope. 

(b)  Osmic  Acid  Test. — Treat  a  small  portion  with  osmic 
acid.     What  happens? 

(c)  Acrolein  Test. — Make  the  acrolein  test  according  to 
directions  on  page  100. 

(d)  "Fusion"  Test  for  Phosphorus. — Place  some  of  the 
lecithin  prepared  above,  in  a  small  porcelain  crucible,  add  a 
suitable  amount  of  a  fusion  mixture  composed  of  KOH  and 
KN03(5:i)  and  heat  carefully  until  the  resulting  mixture 
is  colorless.  Cool,  dissolve  the  mass  in  a  little  warm  water, 
acidify  with  HNO:!,  heat  to  boiling  and  add  a  few  cubic 
centimeters  of  molybdic  solution.  In  the  presence  of  phos- 
phorus a  yellow  precipitate  forms.     What  is  it? 

2.  Preparation  of  Cholesterin. — Place  a  small  amount  of 
macerated  brain  tissue  under  ether  and  stir  occasionally  for 
one  hour.  Filter,  exaporate  the  filtrate  to  dryness  on  a  water- 
bath  and  test  the  cholesterin  according  to  directions  given 
below.  (If  it  is  desired,  the  ether  extract  from  the  so-called 
protagon,  or  the  ether-acetone  filtrate  from  the  lecithin  may 
be  used  for  the  isolation  of  cholesterin.  In  these  cases  it  is 
simply  necessary  to  evaporate  the  solution  to  dryness  on  a 
water-bath.)  Upon  the  cholesterin  prepared  by  either  of  the 
above  methods  make  the  following  tests : 

(a)  Microscopical  Examination. — Examine  the  crystals 
under  the  microscope  and  compare  them  with  those  in  Fig.  42, 
page  125. 

(b)  Iodine-Sulphuric  Acid  Test. — Place  a  few  crystals  of 
cholesterin  in  one  of  the  depressions  of  a  test-tablet  and  treat 
with  a  drop  of  concentrated  sulphuric  acid  and  a  drop  of  a 
very  dilute  solution  of  iodine.  A  play  of  colors,  consisting  of 
violet,  blue,  green  and  red,  results. 


224  PHYSIOLOGICAL    CHEMISTRY. 

(c)  The  Liebermann-Bur  chard  Test. — Dissolve  a  few  crys- 
tals of  cholesterin  in  2  c.c.  of  chloroform  in  a  dry  test-tube. 
Now  add  10  drops  of  acetic  anhydride  and  1-3  drops  of  con- 
centrated sulphuric  acid.  The  solution  becomes  red,  then 
blue,  and  finally  bluish-green  in  color. 

(d)  Salkowski's  Test. — Dissolve  a  few  crystals  of  choles- 
terin in  a  little  chloroform  and  add  an  equal  volume  of  con- 
centrated sulphuric  acid.  A  play  of  colors  from  bluish-red 
to  cherry-red  and  purple  is  noted  in  the  chloroform,  while  the 
acid  assumes  a  marked  green  fluorescence. 

(e)  Schiff's  Reaction. — To  a  little  cholesterin  in  an  evapor- 
ating dish  add  a  few  drops  of  Schiff's  reagent.1  Evaporate 
to  dryness  over  a  low  flame  and  observe  the  reddish-violet 
residue  which  changes  to  a  bluish-violet. 

(/)  Phosphorus. — Test  for  phosphorus  according  to  direc- 
tions given  on  page  223.     Is  phosphorus  present? 

3.  Preparation  of  Cerebrin. — Treat  the  macerated  brain 
tissue,  in  a  flask,  with  95  per  cent  alcohol  and  boil  on  a  water- 
bath  for  one-half  hour,  keeping  the  volume  constant  by  adding 
fresh  alcohol  as  needed.  Filter  the  solution  hot  and  stand 
the  cloudy  filtrate  away  for  twenty-four  hours.  (If  the  fil- 
trate is  not  cloudy  concentrate  it  upon  the  water-bath  until 
it  is  so. )     Filter  off  the  cerebrin  and  test  it  as  follows : 

(a)  Microscopical  Examination. — Suspend  a  small  portion 
in  a  drop  of  water  on  a  slide  and  examine  under  the  micro- 
scope. 

(b)  Solubility. — Try  the  solubility  of  cerebrin  in  the  usual 
solvents  and  in  hot  and  cold  alcohol  and  hot  and  cold  ether. 

(c)  Phosphorus. — Test  for  phosphorus  according  to  direc- 
tions on  page  223.  How  does  the  result  compare  with  that 
on  lecithin. 

(d)  Place  a  little  cerebrin  on  platinum  foil  and  warm. 
Note  the  odor. 

(<?)   Hydrolysis  of  Cerebrin. — Place  the  remaining  cerebrin 

1  Schiff's  reagent  consists  of  a  mixture  of  three  volumes  of  concentrated 
sulphuric  acid  and  one  volume  of  10  per  cent  ferric  chloride. 


NERVOUS    TISSUE.  225 

in  a  small  evaporating  dish,  add  equal  volumes  of  water  and 
dilute  hydrochloric  acid  and  boil  for  one  hour.  Cool,  neutral- 
ize with  solid  potassium  hydroxide,  filter,  and  test  with  Feh- 
ling's  solution.  Is  there  any  reduction,  and  if  so  how  do  you 
explain  it? 


16 


CHAPTER   XVI. 

URINE:  GENERAL  CHARACTERISTICS  OF  NOR- 
MAL AND  PATHOLOGICAL  URINE. 

Volume. — The  volume  of  urine  excreted  by  normal  individ- 
uals, during  any  definite  period,  fluctuates  within  very  wide 
limits.  The  average  output  for  twenty-four  hours  is  placed 
by  German  writers  between  1,500  and  2,000  c.c.  This  value 
is  not  strictly  applicable  to  conditions  in  America  however 
since  it  has  been  found  that  the  average  normal  excretion  of 
the  adult  male  American  falls  within  the  lower  values  of 
1,000-1,200  c.c.  The  volume-excretion  is  influenced  greatly 
by  the  diet,  particularly  by  the  ingestion  of  fluids. 

Certain  pathological  conditions  cause  the  output  of  urine 
for  any  definite  period  to  depart  very  decidedly  from  the  nor- 
mal output.  Among  the  pathological  conditions  in  which  the 
volume  of  urine  is  increased  above  normal  are  the  following: 
Diabetes  mellitus,  diabetes  insipidus,  certain  diseases  of  the 
nervous  system,  contracted  kidney,  amyloid  degeneration  of 
the  kidney  and  in  convalescence  from  acute  diseases  in  gen- 
eral. Many  drugs  such  as  calomel,  digitalis,  acetates  and 
salicylates  also  increase  the  volume  of  the  urine  excreted.  A 
decrease  from  the  normal  is  observed  in  the  following  patho- 
logical conditions :  Acute  nephritis,  diseases  of  the  heart  and 
lungs,  fevers,  diarrhoea  and  vomiting. 

Color. — Normal  urine  ordinarily  possesses  a  yellow  tint, 
the  depth  of  the  color  being  dependent  in  part  upon  the 
density  of  the  fluid.  The  color  of  normal  urine  is  due  prin- 
cipally to  a  pigment  called  urochrome:  traces  of  hcematopor- 
phyrin,  urobilin  and  uroerythrin,  have  also  been  detected. 
Under  pathological  conditions  the  urine  is  subject  to  pro- 
nounced variations  in  color  and  may  contain  many  varieties 
of  pigments.  Under  such  circumstances  the  urine  may  vary 
in  color  from  an  extremely  light  yellow  to  a  very  dark  brown 

226 


URINE. 


227 


or  black.  Vogel  has  constructed  a  color  chart  which  is  of 
some  value  for  purposes  of  comparison.  The  nature  and 
origin  of  the  chief  variations  in  the  urinary  color  are  set  forth 
in  tabular   form  by  Halliburton  as   follows : 


Color. 

Cause  of  Coloration. 

Pathological  Condition. 

Nearly  colorless. 

Dilution,  or  diminution  of 
normal  pigments. 

Increase  of  normal,  or  oc- 
currence of  pathological, 
pigments. 

Nervous    conditions :     hy- 
druria,    diabetes    insipi- 
dus, granular  kidney. 

Dark     yellow     to 
brown-red. 

Acute  febrile  diseases. 

Milky. 

Fat  globules. 
Pus  corpuscles. 

Chyluria. 

Purulent    diseases    of    the 
urinary  tract. 

Orange. 

Excreted  drugs. 

Santonin,     chrysophanic 
acid. 

Red  or  reddish. 

Haematoporphyrin. 

Unchanged  haemoglobin. 

Haemorrhages,    or    haemo- 
globinuria. 

Brown   to   brown- 

Pigments    in    food     (log- 
wood, madder,  bilberries, 
fuchsin). 

Haematin. 

Small  haemorrhages. 

black. 

Methaemoglobin. 

Methaemoglobinuria. 

Melanin. 

Melanotic  sarcoma. 

Hydrochinon  and  catechol. 
Bile-pigments. 

Carbolic-acid  poisoning. 

Greenish-yellow, 
greenish-brown, 
approaching 
black. 

Jaundice. 

Dirty    green1    or 
blue. 

Brown-yellow    to 
red-brown,    be- 
coming    blood- 
red     upon     add- 
ing alkalis. 

A  dark-blue  scum  on  sur- 
face, with  a  blue  deposit, 
due  to  an  excess  of  indi- 
go-forming     substances. 

Substances     contained     in 
senna,  rhubarb,  and   che- 
lidonium   which   are   in- 
troduced   into    the    sys- 
tem. 

Cholera,  typhus ;  seen  espe- 
cially when  the  urine  is 
putrefying. 

1  This  dirty  green  or  blue  color  also  occurs  after  the  use  of  methylene- 
blue  in  the  organism. 


2  28  PHYSIOLOGICAL    CHEMISTRY. 

Transparency. — Normal  urine  is  ordinarily  perfectly  clear 
and  transparent  when  voided.  On  standing  for  a  variable 
time,  however,  a  cloud  (nubecula)  consisting  principally  of 
nucleo-proteid  or  mucoid  (see  p.  264)  and  epithelial  cells 
forms.  A  turbidity  due  to  the  precipitation  of  phosphates  is 
normally  noted  in  urine  passed  after  a  hearty  meal.  The 
urine  obtained  2-3  hours  after  a  meal  or  later  is  ordinarily 
free  from  turbidity.  Permanently  turbid  urines  ordinarily 
arise  from  pathological  conditions. 

Odor. — The  odor  of  normal  urine  is  of  a  faint,  aromatic 
type.  The  bodies  to  which  this  odor  is  due  are  not  well  known, 
but  it  is  claimed  by  some  investigators  to  be  due,  at  least  in 
part,  to  the  presence  of  minute  amounts  of  certain  volatile 
organic  acids.  When  the  urine  undergoes  decomposition,  e.  g., 
in  alkaline  fermentation  a  very  unpleasant  ammoniacal  odor 
is  evolved.  All  urines  are  subject  to  such  decomposition  if 
allowed  to  stand  for  a  sufficiently  long  time.  Under  normal 
conditions  the  urine  very  often  possesses  a  peculiar  odor  due 
to  the  ingestion  of  some  certain  drug  or  vegetable.  For 
instance,  cubebs,  copaiba,  myrtol,  saffron,  tolu  and  turpentine 
each  imparts  a  somewhat  specific  odor  to  the  urine.  After 
the  ingestion  of  asparagus,  the  urine  also  possesses  a  typical 
odor. 

Frequency  of  Urination. — The  frequency  of  urination 
varies  greatly  in  different  individuals  but  in  general  is  de- 
pendent upon  the  amount  of  fluid  in  the  bladder.  In  patho- 
logical conditions  an  inflammatory  affection  of  the  urinary 
tract  or  any  disturbance  of  the  innervation  of  the  bladder  will 
influence  the  frequency.  Affections  of  the  spinal  cord  which 
lead  to  an  increased  irritability  of  the  bladder  or  a  weakening 
of  the  sphincter  will  result  in  increasing  the  frequency  of 
urination. 

Reaction. — The  mixed  twenty-four  hour  urinary  excretion 
of  a  normal  individual  ordinarily  possesses  an  acid  reaction 
to  litmus.  This  acidity  is  now  believed  to  be  due  to  the  pres- 
ence of  various  acidic  radicals  and  not  to  the  presence  of 


URINE. 


229 


sodium  dirhydrogen  phosphate  as  was  formerly  held  (see 
Phosphates,  p.  275).  The  acidity  imparted  to  the  urine  by 
any  particular  acid  depends  entirely  upon  the  extent  to  which 


Fig.  81. 


Deposit  in  Ammoniacal  Fermentation. 
a,   Acid   ammonium    urate;    b,   ammonium    magnesium    phosphate;    c,   bacteria. 


V 


SET 


I* 


«*%  ^ 


Deposit    in    Acid    Fermentation. 
a,  Fungus ;  fc,  amorphous  sodium  urate ;  c,  uric  acid ;  d,  calcium  oxalate. 

the  acid  is  dissociable,  since  it  is  the  hydrogen  ion  which  is 
responsible  for  the  acid  reaction. 

The  composition  of  the  food  is  perhaps  the  most  important 


23O.  PHYSIOLOGICAL    CHEMISTRY. 

factor  in  determining  the  reaction  of  the  urine.  The  reaction 
ordinarily  varies  considerably  according  to  the  time  of  day 
the  urine  is  passed.  For  instance  for  a  variable  length  of  time 
after  a  meal  the  urine  may  be  neutral  or  even  alkaline  in  re- 
action to  litmus,  owing  to  the  claim  of  the  gastric  juice  upon 
the  acidic  radicals  to  further  the  formation  of  hydrochloric 
acid  for  use  in  carrying  out  the  digestive  secretory  function. 
This  change  in  reaction  is  known  as  the  alkaline  tide  and  is 
common  to  perfectly  healthy  individuals.  The  urine  may  also 
become  temporarily  alkaline  in  reaction  to  litmus,  as  the  result 
of  ingesting  alkaline  carbonates  or  certain  salts  of  tartaric  and 
citric  acids  which  may  be  transformed  into  carbonates  within 
the  organism.  Normal  urine  upon  standing  for  some  time 
becomes  alkaline  in  reaction  to  litmus,  owing  to  the  inception 
of  alkaline  or  ammoniacal  fermentation  through  the  agency 
of  micro-organisms.  This  fermentation  has  no  especial  diag- 
nostic value  except  in  cases  where  the  urine  has  undergone  this 
change  within  the  organism  and  is  voided  in  the  decomposed 
state.  Ammoniacal  fermentation  is  ordinarily  due  to  cystitis 
or  occurs  as  the  result  of  infection  in  the  process  of  catheteriza- 
tion. A  microscopical  examination  of  such  urine  (Fig.  81, 
p.  229)  shows  the  presence  of  ammonium  magnesium  phos- 
phate crystals,  amorphous  phosphates  and  not  infrequently 
ammonium  urate. 

Occasionally  a  urine  which  possesses^  normal  acidity  when 
voided,  upon  standing  instead  of  undergoing  ammoniacal  fer- 
mentation as  above  described  will  become  still  more  strongly 
acid  in  reaction.  Such  a  phenomenon  is  termed  acid  fermenta- 
tion. Accompanying  this  increased  acidity  there  is  ordinarily 
a  deepening  of  the  tint  of  the  urinary  color.  Such  urines  may 
contain  acid  urates,  uric  acid,  fungi  and  calcium  oxalate  (Fig. 
82,  p.  229).  On  standing  for  a  sufficiently  long  time  any  urine 
which  exhibits  acid  fermentation  will  ultimately  change  in  re- 
action, due  to  the  inception  of  alkaline  fermentation,  and  will 
show  the  microscopical  deposits  characteristic  of  such  a  urine. 

Specific  Gravity. — The  specific  gravity  of  the  urine  of 
normal  individuals  varies  ordinarily  between  1.015  and  1.025. 


URINE. 


Fig.  83. 


fM 


This  value  is  subject  to  wide  fluctuations  under  various  con- 
ditions. For  instance  following  copious  water-  or  beer-drink- 
ing- the  specific  gravity  may  fall  to  1.003  or  lower,  whereas  in 
cases  of  excessive  perspiration  it  may  rise  as  high  as  1.040 
or  even  higher.  Where  a  very  accurate  de- 
termination of  the  specific  gravity  is  desired 
use  is  commonly  made  of  the  pyknotneter  or  of 
the  Westphal  hydrostatic  balance.  These  in- 
struments, however,  are  not  suited  for  clinical 
use.  The  clinical  method  of  determining  the 
specific  gravity  is  by  means  of  a  urinometer 
(  Fig.  83,  p.  231).  This  affords  a  very  rapid 
method  and  at  the  same  time  is  sufficiently  ac- 
curate for  clinical  purposes.  The  urinometer 
is  always  calibrated  for  use  at  a  specific  tem- 
perature and  the  observations  made  at  any 
other  temperature  must  be  subjected  to  a  cer- 
tain correction  to  obtain  the  true  specific  grav- 
ity. In  makin""  this  correction  one  unit  of  the 
last  order  is  added  to  the  observed  specific 
gravity  for  every  three  degrees  above  the 
normal  temperature  and  subtracted  for  every 
three  degrees  below  the  normal  temperature. 
For  instance,  if  in  using  a  urinometer  cali- 
brated for  1 50  C.  the  specific  gravity  of  a 
urine  having  a  temperature  of  21  °  C.  is  de- 
termined as  1.018  it  is  necessary  to  add  to  the 
observed  specific  gravity  two  units  of  the  third    Urinometer  and 

,  ...  ,  .,,  ...  CYLINDER. 

order  to  obtain  the  real  specific  gravity  ot  the 
urine.     Therefore  the  true  specific  gravity,  at   150   C,  of  a 
urine  having  a  specific  gravity  of  T.018  at  210  C.  is  1.018-f- 
0.002  =  1.020. 

Pathologically,  the  specific  gravity  may  be  subjected  to  very 
wide  variations.  This  is  especially  true  in  diseases  of  the  kid- 
neys. In  acute  nephritis  ordinarily  the  urine  is  concentrated 
and  of  a  high  specific  gravity  whereas  in  chronic  nephritis  the 


232  PHYSIOLOGICAL    CHEMISTRY. 

reverse  conditions  are  more  apt  to  prevail.  In  fact  under  most 
conditions,  whether  physiological  or  pathological,  the  specific 
gravity  of  the  urine  is  inversely  proportional  to  the  volume 
excreted.  This  is  not  true  of  diabetes  mellitus,  however,  where 
the  volume  of  urine  is  large  and  the  specific  gravity  is  also 
high,  owing  to  the  sugar  contained  in  the  urine. 

The  amount  of  solids  eliminated  in  the  excretion  for  twenty- 
four  hours  may  be  roughly  calculated  by  means  of  Long's 
Coefficient,  i.  e.,  2.6.  The  solid  content  of  1,000  c.c.  of  urine 
is  obtained  by  multiplying  the  last  two  figures  of  the  specific 
gravity  observed  at  25 °  C.  by  2.6.  To  determine  the  amount 
of  solids  excreted  in  twenty-four  hours  if  the  volume  was 
1,120  c.c.  and  the  specific  gravity  was  1.018  the  calculation 
,  would  be  as  follows  : 

(a)  18  X  2.6  =  46.8  grams  of  solid  matter  in  1,000  c.c.  of 
urine. 

,,,.  46.8  X  1 120  .    ,.. 

\P) =52.4  grams  of  solid  matter  in  1,120  c.c. 

of  urine. 

The  coefficient  of  Haser  (2.33)  which  has  been  in  use  for 
years  probably  gives  values  that  are  inaccurate  for  conditions 
existing  in  America. 

Freezing-Point  (Cryoscopy). — The  freezing-point  of  a 
solution  depends  upon  the  total  number  of  molecules  of  solid 
matter  dissolved  in  it.  The  determination  of  the  osmotic  pres- 
sure by  this  method  has  recently  come  to  be  of  some  clinical 
importance  particularly  as  an  aid  in  the  diagnosis  of  kid- 
ney disorders.  In  this  connection  it  is  best  to  collect  the 
urine  from  each  kidney  separately  and  determine  the  freezing- 
point  in  the  individual  samples  so  collected.  By  this  means 
considerable  aid  in  the  diagnosis  of  renal  diseases  may  be 
secured.  The  fluids  most  frequently  examined  cryoscopically 
are  the  blood  (see  p.  148)  and  the  urine.  The  freezing-point 
is  denoted  by  A.  The  value  of  A  for  normal  urine  varies  ordi- 
narily between  —  1.30  and  — 2.30  C,  the  freezing-point  of 
pure  water  being  taken  as  o°.     A  is  subject  to  very  wide  flue- 


URINE. 


2  33 


tuations  under  unusual  conditions.  For 
instance  following  copious  water-  or 
beer-drinking  -^  may  have  as  high  a 
value  as  0.2  C.  whereas  on  a  diet 
containing  much  salt  and  deficient  in 
fluids  the  value  of  a  may  be  lowered 
to  — 3  C.  or  even  lower.  The  freez- 
ing-point "t"  normal  blood  is  generally 
about  O.560  C.  and  it  not  subject 
to  the  wide  variations  noted  in  the 
mine,  because  of  the  tendency  of  the 
organism  to  maintain  the  normal  os- 
motic pressure  of  the  blood  under  all 
c< >nditi< >ns.  Variations  between  —  0.5  1 
and  — 0.62 °  C.  may  be  due  entirely 
to  dietary  conditions  but  if  any  marked 
variation  is  noted  it  can,  in  most 
cases,  be  traced  to  a  disordered  kidney 
function. 

Freezing-point  determinations  may 
be  made  by  means  of  the  Beckmann- 
Ileidenhain  apparatus  (Fig.  84.  p.  233  ) 
or  the  Zikel  Pektoscope.  The  Beck- 
mann-Heidenhain  apparatus  consists 
of  the  following  parts :  A  strong  bat- 
ter}- jar  or  beaker  (C)  furnished  with 
a  metal  cover  which  is  provided  with 
a  circular  hole  in  its  center.  This 
strong  glass  vessel  serves  to  hold  the 
freezing  mixture  by  means  of  which 
the  temperature  of  the  fluid  under  ex- 
amination is  lowered.  A  large  glass 
tube  (B)  designed  as  an  air-jacket, 
and  formed  after  the  manner  of  a 
test-tube  is  introduced  through  the 
central    aperture    in    the    metal    cover 


Fig.  84. 


1 


Beckmanm  -  Heiden  MAIN 
Freezing-poj  nt  Ap- 
paratus.      (Long.) 
D,   a   delicate    thermom- 
eter ;     C,     the     containing 
jar;  B,  the  outside  or  air 
mantle   tube;   A,   the   tube 
in    which    the    mixture    to 
be      observed      is      placed. 
Two    stirrers    are    shown, 
one    for   the   cooling    mix- 
ture   in    the    jar    and    one 
for  the  experimental  mix- 
ture. 


234  PHYSIOLOGICAL    CHEMISTRY. 

and  into  this  air-jacket  is  lowered  a  smaller  tube  (A)  con- 
taining the  fluid  to  be  tested.  A  very  delicate  thermom- 
eter (D),  graduated  in  hundredths  of  a  degree  is  intro- 
duced into  the  inner  tube  and  is  held  in  place  by  means  of  a 
cork  so  that  the  mercury  bulb  is  immersed  in  the  fluid  under 
examination  but  does  not  come  in  contact  with  any  glass  sur- 
face. A  small  platinum  wire  stirrer  serves  to  keep  the  fluid 
under  examination  well  mixed  while  a  larger  stirrer  is  used  to 
manipulate  the  freezing  mixture.  (Rock  salt  and  ice  in  the 
proportion  I  13  form  a  very  satisfactory  freezing  mixture.) 

In  making  a  determination  of  the  freezing-point  of  a  fluid 
by  means  of  the  Beckmann-Heidenhain  apparatus  proceed  as 
follows :  Place  the  freezing  mixture  in  the  battery  jar  and  add 
water  (if  necessary)  to  secure  a  temperature  not  lower  than 
3°  C.  Introduce  the  fluid  to  be  tested  into  tube  A,  place  the 
thermometer  and  platinum  wire  stirrer  in  position  and  insert 
the  tube  into  the  air  jacket  which  has  previously  been  inserted 
through  the  metal  cover  of  the  battery  jar.  Manipulate  the 
two  stirrers  in  order  to  insure  an  equalizataion  of  temperature 
and  observe  the  course  of  the  mercury  column  of  the  ther- 
mometer very  carefully.  The  mercury  will  gradually  fall  and 
this  gradual  lowering  of  the  temperature  will  be  followed  by 
a  sudden  rise.  The  point  at  which  the  mercury  rests  after 
this  sudden  rise  is  the  freezing-point.  This  rise  is  due  to  the 
fact  that  previous  to  freezing,  a  fluid  is  always  more  or  less 
over  cooled  and  the  thermometer  temporarily  registers  a  tem- 
perature somewhat  below  the  freezing-point.  As  the  fluid 
freezes  however  there  is  a  very  sudden  change  in  the  tem- 
perature of  the  liquid  and  this  change  is  imparted  to  the  ther- 
mometer and  causes  the  rise  as  indicated.  It  occasionally 
occurs  that  the  fluid  under  examination  is  very  much  over 
cooled  and  does  not  freeze.  Under  such  circumstances  a  small 
piece  of  ice  is  introduced  into  it  by  means  of  the  side  tube 
noted  in  the  figure.  This  so-called  "  inoculation  "  causes  the 
fluid  to  freeze  instantaneously.     (For  details  of  the  method 


URINE.  235 

of  determining  tin-  freezing-point  consult  standard  works  on 
physical  or  organic  chemistry.) 

Electrical  Conductivity. — The  electrical  conductivity  of 
the  urine  is  dependent  up  »n  the  number  of  inorganic  molecules 
« »r  ions  present,  and  in  this  differs  from  the  freezing-point 
which  is  dependent  upon  the  total  number  of  molecules  both 
inorganic  and  organic  which  are  in  solution.  The  conductivity 
of  the  urine  has  been  investigated  but  slightly,  and  this  very 
recently,  but  from  the  data  secured  it  seems  that  the  value 
generally  falls  below  ^  =  0.03.  The  conductivity  of  blood 
serum  has  been  determined  as  «  =0.012.  Up  to  the  present 
time  the  determination  of  the  electrical  conductivity  of  any 
of  the  fluids  of  the  body  has  been  put  to  very  slight  clinical 
use.  Experience  may  show  the  conductivity  value  to  be  a  more 
important  aid  to  diagnosis  than  it  is  now  considered  particu- 
larly if  it  is  taken  in  connection  with  the  determination  of  the 
freezing-point.  By  a  combination  of  these  two  methods  the 
portion  of  the  osmotic  pressure  due  respectively  to  electrolytes 
and  non-electrolytes  may  be  determined.  For  a  discussion  of 
electrical  conductivity,  the  method  by  which  it  is  determined 
and  the  principles  involved  consult  standard  works  on  physical 
or  electrochemistry. 

Collection  of  the  Urine  Sample. — If  any  dependable  data 
are  desired  regarding  the  quantitative  composition  of  the  urine 
the  examination  of  the  mixed  excretion  for  twenty-four  hours 
is  absolutely  necessary.  In  collecting  the  urine  the  bladder 
may  be  emptied  at  a  given  hour,  say  8  A.  M..  the  urine  dis- 
carded and  all  the  urine  from  that  hour  up  to  and  including 
that  passed  the  next  day  at  8  A.  M.  saved,  thoroughly  mixed 
and  a  sample  taken  for  analysis.     Powdered  thymol, 

CH, 

A 

OoH        ■ 

CH3  — CH  — CH3, 


236  PHYSIOLOGICAL    CHEMISTRY. 

is  a  very  satisfactory  preservative  since  the  excess  may  be  re- 
moved by  filtration,  if  desired,  and  any  small  amount  which 
may  go  into  solution  will  have  no  appreciable  influence  upon 
the  determination  of  any  of  the  urinary  constituents.  It  has 
no  reducing  power  and  so  may  safely  be  used  to  preserve  dia- 
betic urines.  To  insure  the  preservation  of  the  mixed  urine  of 
the  twenty- four  hour  period  it  is  advisable  to  place  a  small 
amount  of  the  thymol  powder  in  the  urine  receptacle  before 
the  first  fraction  of  urine  is  voided. 

In  certain  pathological  conditions  it  is  desirable  to  collect 
the  urine  passed  during  the  day  separately  from  that  passed 
during  the  night.  When  this  is  done  the  urine  voided  between 
8  A.  M.  and  8  P.  M.  may  be  taken  as  the  day  sample  and  that 
voided  between  8  P.  M.  and  8  A.  M.  as  the  night  sample. 

The  qualitative  testing  of  urine  voided  at  random,  except 
in  a  few  specific  instances,  is  of  no  particular  value  so  far  as 
giving  us  any  accurate  knowledge  as  to  the  exact  urinary 
characteristics  of  the  individual  is  concerned.  In  the  great 
majority  of  cases  the  qualitative  as  well  as  the  quantitative 
tests  should  be  made  upon  the  mixed  excretion  for  a  twenty- 
four  hour  period. 


CHAPTER    XVII. 


URINE:    PHYSIOLOGICAL    CONSTITUENTS. 


i.  Organic  Physiological  Constituents. 


Urea. 
I'ric  acid. 
Creatinin. 


Ethereal   sulphuric   acids. 


Hippuric  acid. 
(  ).\alic  acid. 


Neutral  sulphur  compounds. 


Allantoin. 


Aromatic  oxvacids - 


Phenol-  and  />-cresol-sulphuric 

acids. 

Pyrocatechin-sulphuric  acid. 
Indoxyl-sulphuric  acid. 
Skatoxyl-sulphuric  acid. 


Cystin. 

Chondroitin-sulphuric  acid. 
Sulphocyanides. 
Taurin  derivatives. 
Oxyproteic  acid. 
Alloxyproteic  acid. 
Uroferric  acid. 

I  \araoxyphenyl-acetic  acid. 
Paraoxyphenyl-propionic  acid. 
Homogentisic  acid. 
Uroleucic  acid. 
Oxymandelic  acid. 
Kvnurenic  acid. 


Benzoic  acid. 
Xucleo-proteid. 
Oxaluric  acid. 

1  It  is  impossible  to  make  any  absolute  classification  of  the  physiological 
and  pathological  constituents  of  the  urine.  A  substance  may  be  present  in 
the  urine  in  small  amount  physiologically  and  be  sufficiently  increased 
under  certain  conditions  as  to  be  termed  a  pathological  constituent. 
Therefore  it  depends,  in  some  instances,  upon  the  quantity  of  a  constituent 
present  whether  it  may  be  correctly  termed  a  physiological  or  a  patholog- 
ical constituent. 

^37 


238  PHYSIOLOGICAL    CHEMISTRY. 

Enzymes    i?:Psil\  ,.       ,       N 

[Diastatic  enzyme  (Amylase), 

f  Acetic  acid. 
Volatile  fatty  acids -I  Butyric  acid. 

L  Formic  acid. 

Paralactic  acid. 

Phenaceturic  acid. 

Phosphorized  compounds.  .  .  ( Glvcerophosphoric  acid. 
1  t  Phospnocarnic  acid. 

fUrochrome. 

Pigments 1  Urobilin. 

L  Uroerythrin. 


Ptomaines  and  leucomaines. 


Purin  bases 


'  Adenin. 

Guanin. 

Xanthin. 

Epiguanin. 

Episarkin. 

Hypoxanthin. 

Paraxanthin. 

Heteroxanthin. 
.  i-Methylxanthin. 


2.  Inorganic  Physiological  Constituents. 
Ammonia. 
Sulphates. 
Chlorides. 
Phosphates. 

Sodium  and  potassium. 
Calcium  and  magnesium. 
Carbonates. 
Iron. 

Fluorides. 
Nitrates. 
Silicates. 
Hydrogen  peroxide. 


URINE.  239 

I 

UREA,  (  j  =  O. 
I 

NH, 

Urea  is  the  principal  end-product  of  the  metabolism  of  pro- 
teid  bodies.  It  has  been  generally  believed  that  about  90  per 
cent  of  the  total  nitrogen  of  the  urine  was  present  as  urea. 
Recently,  however,  Folin  has  shown  that  the  distribution  of  the 
nitrogen  of  the  urine  among  urea  and  the  other  nitrogen- 
containing  bodies  present  depends  entirely  upon  the  absolute 
amount  of  the  total  nitrogen  excreted.  He  found  that  a  de- 
crease in  the  total  nitrogen  excretion  was  always  accompanied 
by  a  decrease  in  the  percentage  of  the  total  nitrogen  excreted 
as  urea,  and  that  after  so  regulating  the  diet  of  a  normal  per- 

Fig.  85. 


Urea. 

son  as  to  cause  the  excretion  of  total  nitrogen  to  be  reduced 
to  3-4  grams  in  24  hours,  only  about  60  per  cent  of  this  nitro- 
gen appeared  in  the  urine  as  urea.  His  experiments  also  seem 
to  show  urea  to  be  the  only  one  of  the  nitrogenous  excretions 
which  is  relatively  as  well  as  absolutely  decreased  as  a  result 


24O  PHYSIOLOGICAL    CHEMISTRY. 

of  decreasing  the  amount  of  proteid  metabolized.  This  same 
investigator  reports  a  hospital  case  in  which  only  14.7  per  cent 
of  the  total  nitrogen  was  present  as  urea  and  about  40  per 
cent  was  present  as  ammonia.  Morner  had  previously  reported 
a  case  in  which  but  4.4  per  cent  of  the  total  nitrogen  of  the 
urine  was  present  as  urea,  and  26.7  per  cent  was  present  as 
ammonia. 

Urea  occurs  most  abundantly  in  the  urine  of  man  and  car- 
nivora  and  in  somewhat  smaller  amount  in  the  urine  of  herbi- 
vora;  the  urine  of  fishes,  amphibians  and  certain  birds  also 
contains  a  small  amount  of  the  substance.  Urea  is  also  found 
in  nearly  all  the  fluids  and  in  many  of  the  tissues  and  organs 
of  mammals.  The  amount  excreted  under  normal  conditions, 
by  an  adult  man  in  24  hours  is  about  30  grams ;  women  excrete 
a  somewhat  smaller  amount.  The  excretion  is  greatest  in 
amount  after  a  diet  of  meat,  and  least  in  amount  after  a  diet 
consisting  of  non-nitrogenous  foods;  this  is  due  to  the  fact 
that  the  last  mentioned  diet  has  a  tendency  to  decrease  the 
metabolism  of  the  tissue  proteids  and  thus  cause  the  output 
of  urea  under  these  conditions  to  fall  below  the  output  of  urea 
observed  during  starvation.  The  output  of  urea  is  also  in- 
creased after  copious  water-  or  beer-drinking.  This  increase 
is  due  primarily  to  the  washing  out  of  the  tissues  of  the  urea 
previously  formed,  but  which  had  not  been  removed  in  the  nor- 
mal processes,  and,  secondarily  to  a  stimulation  of  proteid 
catabolism. 

Urea  may  be  formed  in  the  organism  from  amino  acids 
such  as  leucin,  glycocoll  and  aspartic  acid:  it  may  also  be 
formed  from  ammonium  carbonate  (NH4)2C03  or  ammonium 
carbamate,  H4N  ■  O  ■  CO  ■  NH2. 

There  are  differences  of  opinion  regarding  the  transforma- 
tion of  the  substances  just  named  into  urea  but  there  is  rather 
conclusive  evidence  that  at  least  a  part  of  the  urea  is  formed 
in  the  liver;  it  maybe  formed  in  other  organs  or  tissues  as  well. 

Urea  crystallizes  in  long,  colorless,  four  or  six-sided,  anhy- 
drous, rhombic  prisms  (Fig.  85,  p.  239),  which  melt  at  1320  C. 
and  are  soluble  in  water  or  alcohol  and  insoluble  in  ether  or 


URINE.  241 

chloroform.  If  a  crystal  of  urea  is  heated  in  a  test-tube,  it 
melts  and  decomposes  with  the  liberation  of  ammonia.  The 
residue  contains  cyanuric  acid, 

A 

N     N 

II       I 

HO  ■  C      0  •  OH 

\// 
N 
and  biuret, 

NH2 

C  =  0 
^>XH 

C  =  0 

I 
NHo. 

The  biuret  may  be  dissolved  in  water  and  a  reddish-violet  color 
obtained  by  treating  the  aqueous  solution  with  cupric  sulphate 
and  potassium  hydroxide  (see  Biuret  Test,  p.  45).  Certain 
hypochlorites  or  hypobromites  in  alkaline  solution  have  the 
power  of  decomposing  urea  into  nitrogen,  carbon  dioxide  and 
water.  Sodium  hypobromite  brings  about  this  decomposition 
as  follows : 

CO(XH2)2  +  3XaOBr  =  3NaBr  +  N2  +  C02  +  2H20. 

This  property  forms  the  basis  for  the  clinical  quantitative 
determination  of  urea   (see  page  35 1  ) . 

Urea  has  the  power  of  forming  crystalline  compounds  with 
certain  acids :  urea  nitrate  and  urea  oxalate  are  the  most  im- 
portant of  these  compounds.  Urea  nitrate,  CO(NH2)2' 
HXOo.  crystallizes  in  colorless,  rhombic  or  six-sided  tiles  (Fig. 
86,  p.  242),  which  are  easily  soluble  in  water.  Urea  oxalate, 
2  •  CO(XH2)2- H2C204,  crystallizes  in  the  form  of  rhombic 
"r  six-sided  prisms  or  plates  (Fig.  88,  p.  244)  :  the  oxalate 
differs  from  the  nitrate  in  being  somewhat  less  soluble  in  water. 
17 


242 


PHYSIOLOGICAL    CHEMISTRY. 


A  decrease  in  the  excretion  of  urea  is  observed  in  many- 
diseases  in  which  the  diet  is  much  reduced  and  in  some  dis- 
orders as  a  result  of  alterations  in  metabolism,  e.  g.}  myxce- 
dema,  and  in  others  as  a  result  of  changes  in  excretion,  as  in 

Fig.  86. 


Urea   Nitrate. 


severe  and  advanced  kidney  disease.  A  pathological  increase 
is  found  in  a  large  proportion  of  diseases  which  are  asso- 
ciated with  a  toxic  state. 

Experiments  on  Urea. 
1.  Isolation  from  the  Urine. — Place  800  c.c.  of  urine  in  a 
precipitating  jar,  add  250  c.c.  of  baryta  mixture1  and  stir  thor- 
oughly. Filter  off  the  precipitate  of  phosphates,  sulphates, 
urates  and  hippurates  and  evaporate  the  filtrate  on  a  water- 
bath  to  a  thick  syrup.  This  syrup  contains  chlorides,  creatinin, 
organic  salts,  pigments  and  urea.  Extract  the  syrup  with 
warm  95  per  cent  alcohol  and  filter  again.  The  filtrate  contains 
the  urea  contaminated  with  pigment.  Decolorize  the  filtrate 
by  boiling  with  animal  charcoal,  filter  again  and  stand  the 

1  Baryta  mixture  consists  of  a  mixture  of  one  volume  of  a  saturated 
solution  of  Ba(N03) 2  and  two  volumes  of  a  saturated  solution  of  Ba (OH) 2. 


URINE. 


243 


Fig.  87. 


filtrate  away  in  a  cold  place  for  crystallization.  Examine  the 
crystals  under  the  microscope  and  compare  them  with  those 
shown  in  Fig.  85,  page  239. 

2.  Solubility. — Test  the  solubility  of  urea,  prepared  by 
yourself  or  furnished  by  the  instructor,  in  the  ordinary  sol- 
vent- (see  p.  4)  and  in  alcohol  and  ether. 

3.  Melting-Point. — Determine  the 
melting  point  of  somepure  urea  furnished 
by  the  instructor.  Proceed  as  follows: 
Into  an  ordinary  melting-point  tube, 
sealed  at  one  end,  introduce  a  crystal  of 
urea.  Fasten  the  tube  to  the  bulb  of  a 
thermometer  as  shown  in  Fig.  87,  p.  243, 
and  suspend  the  bulb  and  its  attached 
tube  in  a  small  beaker  containing  sul- 
phuric acid.  Gently  raise  the  tempera- 
ture of  the  acid  by  means  of  a  low  flame, 
stirring  the  fluid  continually,  and  note  the 
temperature  at  which  the  urea  begins  to 
melt. 

4.  Crystalline  Form.  —  Dissolve  a 
crystal  of  pure  urea  in  a  few  drops  of 
95  per  cent  alcohol  and  place  1-2  drops 
of  the  alcoholic  solution  on  a  microscopic 
slide.  Allow  the  alcohol  to  evaporate 
spontaneously,  examine  the  crystals  under 
the  microscope  and  compare  them  with 
those  reproduced  in  Fig.  85,  p.  239.  Re- 
crystallize  a  little  urea  from  water  in  the 
same  way  and  compare  the  crystals  with 
those  obtained  from  the  alcoholic  solution. 

5.  Formation  of  Biuret. — Place  a  small  amount  of  urea  in 
a  dry  test-tube  and  heat  carefully  in  a  low  flame.  The  urea 
melts  at  1320  C.  and  liberates  ammonia.  Continue  heating 
until  the  fused  mass  begins  to  solidify.  Cool  the  tube,  dis- 
solve the  residue  in  dilute  potassium  hydroxide  'solution  and 
add   very  dilute  cupric  sulphate  solution    (see  p.   45).     The 


Melting-point  Tubes 
Fastened  to  Bui.b  of 
Thermometer. 


244 


PHYSIOLOGICAL    CHEMISTRY. 


purplish-violet  color  is  due  to  the  presence  of  biuret  which  has 
been  formed  from  the  urea  through  the  application  of  heat  as 
indicated.     This  is  the  reaction: 


NH2 
2  C  =  0 

NH2 

Urea. 


NH2 

C  =  0 

\ 

NH  +  NHa 

/ 
C  =  0 

NH2 

Biuret. 


6.  Urea  Nitrate. — Prepare  a  concentrated  solution  of  urea 
by  dissolving  a  little  of  the  substance  in  a  few  drops  of  water. 
Place  a  drop  of  this  solution  on  a  microscopic  slide,  add  a  drop 


Fig. 


Urea  Oxalate. 


of  concentrated  nitric  acid  and  examine  under  the  microscope. 

Compare  the  crystals  with  those  reproduced  in  Fig.  86,  p.  242. 

7.  Urea  Oxalate. — To  a  drop  of  a  concentrated  solution  of 

urea, prepared  as  described  in  the  last  experiment(6),add  a  drop 


URINE.  245 

of  a  saturated  solution  of  oxalic  acid.  Examine  under  the 
microscope  and  compare  the  crystals  with  those  shown  in  Fig. 
88.  page  244. 

8.  Decomposition  by  Sodium-Hypobromite. — Into  a 
mixture  of  3  c.c.  of  concentrated  sodium  hydroxide  solution 
and  2  c.c.  of  bromine  water  in  a  test-tube  introduce  a  crystal 
of  urea  or  a  small  amount  of  a  concentrated  solution  of  urea. 
Through  the  influence  of  the  sodium-hypobromite,  XaOBr, 
the  urea  is  decomposed  and  carbon  dioxide  and  nitrogen  are 
liberated.  The  carbon  dioxide  is  absorbed  by  the  excess  of 
sodium  hydroxide  while  the  nitrogen  is  evolved  and  causes 
the  marked  effervescence  observed.  This  property  forms  the 
basis  for  one  of  the  methods  in  common  use  for  the  quanti- 
tative determination  of  urea.  Write  the  equation  showing 
the  decomposition  of  urea  by  sodium-hypobromite. 

9.  Furfurol  Test. — To  a  few  crystals  of  urea  in  a  small 
porcelain  dish  add  1-2  drops  of  a  concentrated  aqueous  solu- 
tion of  furfurol  and  1-2  drops  of  concentrated  hydrochloric 
acid.  Note  the  appearance  of  a  yellow  color  which  gradually 
changes  into  a  purple.  Allantoin  also  responds  to  this  test 
(see  page  261). 

HX  —  CO 

I         I 
URIC  ACID,  OC       C-  NHV 

I       II  >C0. 

HN  —  C  —  NET 

Uric  acid  is  one  of  the  most  important  of  the  constituents 
of  the  urine.  Normally  about  0.7  gram  is  excreted  in  24  hours 
but  this  amount  is  subject  to  wide  variations,  particularly  under 
certain  dietary  and  pathological  conditions.  Uric  acid  is  a 
diureide  and  consequently  upon  oxidation  yields  two  molecules 
of  urea.  It  acts  as  a  weak  dibasic  acid  and  forms  two  classes  of 
salts,  neutral  and  acid.  The  neutral  potassium  and  lithium 
urates  are  the  most  easily  soluble  of  the  alkali  salts;  the  am- 
monium urate  is  difficultly  soluble.  The  acid-alkali  urates  are 
more  insoluble  and  form  the  major  portion  of  the  sediment 


246  PHYSIOLOGICAL    CHEMISTRY. 

which  separates  upon  cooling  concentrated  urine;  the  alkaline 
earth  urates  are  very  insoluble.  Ordinarily  uric  acid  occurs  in 
the  urine  in  the  form  of  urates  and  upon  acidifying  the  liquid 
the  uric  acid  is  liberated  and  deposits  in  crystalline  form.  This 
property  forms  the  basis  for  one  of  the  older  methods  for  the 
quantitative  determination  of  uric  acid  (Heintz  Method,  p.  350) . 

Uric  acid  is  very  closely  related  to  the  purin  bases  as  may 
be  seen  from  a  comparison  of  its  structural  formula  with 
those  of  the  purin  bases  given  on  page  211.  According  to  the 
purin  nomenclature  it  is  designated  2-6-8-trioxypurin.  Uric 
acid  forms  the  principal  end-product  of  the  nitrogenous  meta- 
bolism of  birds  and  scaly  amphibians ;  in  the  human  organism 
it  occupies  the  fourth  position  inasmuch  as  here  urea,  am- 
monia and  creatinin  are  the  chief  end-products  of  nitrog- 
enous metabolism.  The  relation  existing  between  uric  acid 
and  urea  in  human  urine  under  normal  conditions  varies  on  the 
average  from  1 :  40  to  1:  100  and  is  subject  to  wider  varia- 
tions under  pathological  conditions.  Because  of  the  high 
content  of  uric  acid  in  the  urine  of  new-born  infants  the  ratio 
may  be  reduced  to  1  :  10  or  even  lower. 

In  man,  uric  acid  probably  results  principally  from  the 
destruction  of  nuclein  material.  It  may  arise  from  nuclein 
or  other  purin  material  ingested  as  food  or  from  the  disin- 
tegrating cellular  matter  of  the  organism.  The  uric  acid 
resulting  from  the  first  process  is  said  to  be  of  exogenous 
origin,  whereas  the  product  of  the  second  form  of  activity  is 
said  to  be  of  endogenous  origin.  As  the  result  of  experimen- 
tation, Siven,  and  Burian  and  Schur,  and  Rockwood  claim 
that  the  amount  of  endogenous  uric  acid  formed  in  any  given 
period  is  fairly  constant  for  each  individual  under  normal 
conditions,  and  that  it  is  entirely  independent  of  the  total 
amount  of  nitrogen  eliminated.  Recently  Folin  has  taken  ex- 
ception to  the  statements  of  these  investigators  and  claims 
that,  following  a  pronounced  decrease  in  the  amount  of  pro- 
teid  metabolized,  the  absolute  quantity  of  uric  acid  is  decreased 
but  that  this  decrease  is  relatively  smaller  than  the  decrease 


PLATE   V. 


Uric   Acid  Crystals.     Normal  Color.      (From  Purely,  after  Peyer.) 


URINE.  -}7 

in  the  total  nitrogen  excretion  and  that  the  per  cent  of  the  uric 
acid  nitrogen,  in  terms  of  the  total  nitrogen,  is  therefore  de- 
cidedly increased. 

In  birds  and  scaly  amphibians  the  formation  of  uric  acid 
is  analogous  to  the  formation  of  urea  in  man.  In  these  or- 
ganisms it  is  derived  principally  from  the  proteid  material 
of  the  tissues  and  the  food  and  is  formed  through  a  process 
of  synthesis  which  occurs  for  the  most  part  in  the  liver;  a 
comparatively  small  fraction  of  the  total  uric  acid  excretion  of 
birds  and  scaly  amphibians  may  result  from  nuclein  material. 

When  pure,  uric  acid  may  be  obtained  as  a  white,  odorless, 
and  tasteless  powder  which  is  composed  principally  of  small 
transparent  crystalline  rhombic  plates.  Uric  acid  as  it  sepa- 
rates from  the  urine  is  invariably  pigmented,  and  crystallizes 
in  a  large  variety  of  characteristic  forms,  c.  g.,  dumb-bells, 
wedges,  rhombic  prisms,  irregular  rectangular  or  hexagonal 
plates,  whetstones,  prismatic  rosettes,  etc.  Uric  acid  is  in- 
soluble in  alcohol  and  ether,  soluble  with  difficulty  in  boiling 
water  {i  :  1800)  and  practically  insoluble  in  cold  water 
(1:39.480,  at  i8°C).  It  is  soluble  in  alkalis,  alkali  car- 
bonates, boiling  glycerin,  concentrated  sulphuric  acid  and  in 
certain  organic  bases  such  as  ethylamine  and  piperidin.  It  is 
claimed  that  the  uric  acid  is  held  in  solution  in  the  urine  by 
the  urea  and  di-sodium  hydrogen  phosphate  present.  Uric 
acid  possesses  the  power  of  reducing  cupric  hydroxide  in 
alkaline  solution  and  may  thus  lead  to  an  erroneous  conclusion 
in  testing  for  sugar  in  the  urine  by  means  of  Fehling's  or 
Trommer's  tests.  A  white  precipitate  of  cuprous  urate  is 
formed  if  only  a  small  amount  of  cupric  hydroxide  is  present, 
but  if  enough  of  the  copper  salt  is  present  the  characteristic 
red  or  brownish-red  precipitate  of  cuprous  oxide  is  obtained. 
Uric  acid  does  not  possess  the  power  of  reducing  bismuth  in 
alkaline  solution  and  therefore  does  not  interfere  in  testing  for 
sugar  in  the  urine  by  means  of  Boettger's  or  Xy lander's  tests. 

In  addition  to  being  an  important  urinary  constituent  uric 
acid  is  normally  present  in  the  brain,  heart,  liver,  lungs,  pan- 


248  PHYSIOLOGICAL    CHEMISTRY. 

creas  and  spleen;  it  also  occurs  in  the  blood  of  birds  and  has 
been  detected  in  traces  in  human  blood  under  normal  condi- 
tions. 

Pathologically,  the  excretion  of  uric  acid  is  subject  to  wide 
variations  but  the  experimental  findings  are  rather  contradic- 
tory. It  may  be  stated  with  certainty,  however,  that  in 
leukaemia  the  uric  acid  output  is  increased  absolutely  as  well 
as  relatively  to  the  urea  output;  under  these  conditions  the 
ratio  between  the  uric  acid  and  urea  may  be  as  low  as  1:9, 
whereas  the  normal  ratio,  as  we  have  seen,  is  1 150  or  higher. 
In  the  study  of  the  influence  of  X-ray  on  metabolism  Edsall 
has  very  recently  reached  some  interesting  conclusions.  He 
found  that  the  excretion  of  uric  acid  is  usually  increased  and 
that  in  some  conditions,  particularly  in  leukaemia,  it  may  be 
greatly  increased.  The  excretion  of  total  nitrogen,  phos- 
phates and  other  substances  may  also  be  considerably  increased. 

Experiments  on  Uric  Acid. 

1.  Isolation  from  the  Urine. — Place  about  200  c.c.  of 
filtered  urine  in  a  beaker,  render  it  acid  with  2-10  c.c.  of  con- 
centrated hydrochloric  acid,  stir  thoroughly  and  stand  the 
vessel  in  a  cold  place  for  24  hours.  Examine  the  pigmented 
crystals  of  uric  acid  under  the  microscope  and  compare  them 
with  those  shown  in  Fig.  101,  p.  323  and  PI.  V.  opposite  p.  247. 

2.  Solubility. — Try  the  solubility  of  pure  uric  acid,  fur- 
nished by  the  instructor,  in  the  ordinary  solvents  (see  p.  4) 
and  in  alcohol,  ether,  concentrated  sulphuric  acid  and  in  boiling 
glycerin. 

3.  Crystalline  Form  of  Pure  Uric  Acid. — Place  about  100 
c.c.  of  water  in  a  small  beaker,  render  it  distinctly  alkaline  with 
potassium  hydroxide  solution  and  add  a  small  amount  of  pure 
uric  acid  stirring  continuously.  Cool  the  solution,  render  it 
distinctly  acid  with  hydrochloric  acid  and  allow  it  to  stand 
in  a  cool  place  for  crystallization.  Examine  the  crystals  under 
the  microscope  and  compare  them  with  those  reproduced  in 
Fig.  89,  page  249. 


URINE. 


249 


4.  Murexid  Test. — To  a  small  amount  of  pure  uric  acid 
in  a  small  evaporating  dish  add  2-3  drops  of  concentrated 
nitric  acid.  Evaporate  to  dryness  carefully  on  a  water-bath 
or  over  a  very  low  flame.     A  red  or  yellow  residue  remains 

Fig.  89. 


Pure  Uric   Acid. 

which  turns  purplish-red  after  cooling  the  dish  and  adding  a 
drop  of  very  dilute  ammonium  hydroxide.  The  color  is  due 
to  the  formation  of  murexid.  If  potassium  hydroxide  is  used 
instead  of  ammonium  hydroxide  a  purplish-violet  color  due 
to  the  production  of  the  potassium  salt  is  obtained.  The  color 
disappears  upon  warming;  with  certain  related  bodies  (purin 
bases)  the  color  persists  under  these  conditions. 

5.  Scruff's  Reaction. — Dissolve  a  small  amount  of  pure 
uric  acid  in  sodium  carbonate  solution  and  transfer  a  drop  of 
the  resulting  mixture  to  a  strip  of  filter  paper  saturated  with 
argentic  nitrate  solution.  A  yellowish-brown  or  black  colora- 
tion due  to  the  formation  of  reduced  silver  is  produced. 

6.  Influence  upon  Fehling's  Solution. — Dilute  1  c.c.  of 
Fehling's  solution  with  4  c.c.  of  water  and  heat  to  boiling. 
Xow  add  slowly,  a  few  drops  at  a  time,  1-2  c.c.  of  a  concen- 
trated solution  of  uric  acid  in  potassium  hydroxide,  heating 


250  PHYSIOLOGICAL    CHEMISTRY. 

after  each  addition.  From  this  experiment  what  do  you  con- 
clude regarding  the  possibility  of  arriving  at  an  erroneous 
decision  when  testing  for  sugar  in  the  urine  by  means  of 
Fehling's  test? 

7.  Reduction  of  Nylander's  Reagent. — To  5  c.c.  of  a  solu- 
tion of  uric  acid  in  potassium  hydroxide  add  about  one-half 
a  cubic  centimeter  of  Nylander's  reagent  and  heat  to  boiling 
for  a  few  moments.  Do  you  obtain  the  typical  black  end- 
reaction  signifying  the  reduction  of' the  bismuth? 

NH CO 

CREATININ,  C  =  NH    I 

I  I 

N-CH3-CH2. 

Creatinin  is  the  anhydride  of  creatin  and  is  a  constituent  of 
normal  human  urine.  It  is  derived  from  the  creatin  of  in- 
gested muscular  tissue  as  well  as  from  the  creatin  of  the 
muscular  tissue  of  the  organism.  Under  normal  conditions 
about  1  gram  of  creatinin  is  excreted  by  an  adult  man  in  24 
hours,  the  exact  amount  depending  in  great  part  upon  the 
nature  of  the  food  and  decreasing  markedly  in  starvation. 
Very  little  that  is '  important  is  known  regarding  the  excre- 
tion of  creatinin  under  pathological  conditions.  The  creatinin 
content  of  the  urine  is  said  to  be  increased  in  typhoid  fever, 
typhus,  tetanus  and  pneumonia,  and  to  be  decreased  in  anaemia, 
chlorosis,  paralysis  and  in  advanced  degeneration  of  the  kid- 
neys. The  greater  part  of  the  data,  however,  relating  to  the 
variation  of  the  creatinin  excretion  under  pathological  condi- 
tions are  not  of  much  value  since,  in  nearly  every  instance, 
the  diet  was  not  sufficiently  controlled  to  permit  the  collection 
of  reliable  data. 

Creatinin  crystallizes  in  colorless,  glistening  monoclinic 
prisms  (Fig.  90,  p.  251)  which  are  soluble  in  about  12  parts 
of  cold  water;  they  are  more  soluble  in  warm  water  and  in 
warm  alcohol.  One  of  the  most  important  and  interesting  of 
the     compounds     of     creatinin     is     creatinin-zinc     chloride, 


t'RIXK. 


251 


(C4H7N30)oZnCl1,,  which  is  formed  from  an  alcoholic  solu- 
tion of  creatinin  upon  treatment  with  zinc  chloride  in  acid 
solution.  Creatinin  has  the  power  of  reducing  cupric  hy- 
droxide in  alkaline  solution  and  in  this  way  may  interfere  with 
the  determination  of  sugar  in  the  urine.  In  the  reduction  by 
creatinin  the  blue  liquid  is  first  changed  to  a  yellow  and  the 
formation  of  a  brownish-red  precipitate  of  cuprous  oxide  is 
brought  about  only  after  continuous  boiling  with  an  excess 
of  the  copper  salt.  Creatinin  does  not  reduce  alkaline  bismuth 
solutions  and   therefore  does  not  interfere  with   Nylander's 


and  Boettger's  tests. 


I' ig.  90. 


Creatinin. 


It  has  very  recently  been  shown  by  Folin  that  the  absolute 
quantity  of  creatinin  eliminated  in  the  urine  on  a  meat-free 
diet  is  a  constant  quantity  different  for  different  individuals, 
but  wholly  independent  of  quantitative  changes  in  the  total 


amount  of  nitrogen  eliminated. 


Experiments  on  Creatinin. 
1.  Separation  from  the  Urine. — Place  250  c.c.  of  urine  in 
a  casserole  or  beaker,  render  it  alkaline  with  milk  of  lime  and 
then  add  CaCl2  solution  until  the  phosphates  are  completely 


252 


PHYSIOLOGICAL    CHEMISTRY. 


precipitated.  Filter  off  the  precipitate,  render  the  filtrate 
slightly  acid  with  acetic  acid  and  evaporate  it  to  a  syrup. 
While  still  warm  this  syrup  is  treated  with  about  50  c.c.  of 
95-97  per  cent  alcohol  and  the  mixture  allowed  to  stand  8-12 
hours  in  a  cool  place.  The  precipitate  is  now  filtered  off  and 
the  filtrate  treated  with  a  little  sodium  acetate  and  about  one- 
half  c.c.  of  acid-free  zinc  chloride  solution  having  a  specific 
gravity  of  1.2.  This  mixture  is  stirred  thoroughly  and  allowed 
to  stand  in  a  cold  place  for  48-72  hours.  Creatinin-zinc 
chloride  (Fig.  91,  below)  will  crystallize  out  under  these  con- 

Fig.  91. 


Creatinin-Zinc   Chloride.     (Salkozi'ski.) 


ditions.  Collect  the  crystals  on  a  filter  paper  and  wash  them 
with  alcohol  to  remove  chlorides.  Now  treat  the  zinc  chloride 
compound  with  a  little  warm  water,  boil  with  lead  oxide  and 
filter.  The  filtrate  may  now  be  decolorized  by  animal  charcoal, 
evaporated  to  dryness  and  the  residue  extracted  with  strong 
alcohol.  (Creatin  remains  undissolved  under  these  condi- 
tions.) The  alcoholic  extract  of  creatinin  is  now  evaporated 
to  incipient  crystallization  and  left  in  a  cool  place  until  crys- 
tallization is  complete.  If  desired  the  crystals  may  be  purified 
by  recrystallization  from  water. 

2.  Weyl's  Test. — Take  5  c.c.  of  urine  in  a  test-tube,  add  a 


URINE.  253 

few  drops  of  sodium  nitro-prusside  and  render  the  solution 
alkaline  with  potassium  hydroxide  solution.  A  ruby  red  color 
results  which  soi in  turns  yellow.  See  Legal's  test  for  ace- 
lour,  page  305. 

3.  Salkowski's  Test. — To  the  yellow  solution  obtained  in 
WVyl's  test  above  add  an  excess  of  acetic  acid  and  apply  heat. 
A  green  color  results  and  is  in  turn  displaced  by  a  blue  color. 
A  precipitate  of  Prussian  blue  may  form. 

4.  Jaffe's  Reaction. — Place  5  c.c.  of  urine  in  a  test-tube, 
add  an  aqueous  solution  of  picric  acid  and  render  the  mixture 
alkaline  with  potassium  hydroxide  solution.  A  red  color  is 
produced  which  turns  yellow  if  the  solution  be  acidified.  Dex- 
trose gives  a  similar  red  color  but  only  upon  the  application 
of  heat.  This  color  reaction  observed  when  creatinin  in  alka- 
line solution  is  treated  with  picric  acid  is  the  basic  principle 
of  Folin's  colorimetric  method  for  the  quantitative  determi- 
nation of  creatinin  (see  page  369). 

ETHEREAL  SULPHURIC  ACIDS. 

The  most  important  of  the  ethereal  sulphuric  acids  found 
in  the  urine  are  phenol-sulphuric  acid,  p-crcsol-sulphuric  acid, 
iihiihvyl-sulphitric  acid  and  skatoxyl-sulphuvic  acid.  Pyro- 
catechin-sulphuric  acid  also  occurs  in  traces  in  human  urine. 
The  total  output  of  ethereal  sulphuric  acid  varies  from  0.09 
to  0.62  gram  for  24  hours.  In  health  the  ratio  of  ethereal  sul- 
phuric acid  to  inorganic  sulphuric  acid  is  about  1  :  10.  These 
ethereal  sulphuric  acids  originate  in  part  from  the  phenol, 
cresol,  indol  and  skatol  formed  in  the  putrefaction  of  proteid 
material  in  the  intestine.  The  phenol  passes  into  the  urine 
directly  as  the  corresponding  ethereal  sulphuric  acid  whereas 
the  indol  and  skatol  undergo  a  preliminary  oxidation  to  form 
indoxyl  and  skatoxyl  respectively  before  their  elimination. 

It  has  generally  been  considered  that  each  of  the  ethereal 
sulphuric  acids  was  formed  principally  in  the  putrefaction  of 
proteid  material  in  the  intestine  and  that  therefore  a  determi- 
nation of  the  total  ethereal  sulphuric  acid  content  of  the  urine 
was  an  index  of  the  extent  to  which  these  putrefactive  proc- 


254  PHYSIOLOGICAL    CHEMISTRY. 

esses  were  proceeding-  within  the  organism.  Recently,  how- 
ever, Folin  has  conducted  a  series  of  experiments  which  seem 
to  show  that  the  ethereal  sulphuric  acid  content  of  the  urine 
does  not  afford  an  index  of  the  extent  of  intestinal  putrefac- 
tion,, since  these  bodies  arise  only  in  part  from  putrefactive 
processes.  He  claims  that  the  ethereal  sulphuric  acid  excretion 
represents  a  form  of  sulphur  metabolism  which  is  more  in 
evidence  upon  a  diet  containing  a  very  small  amount  of  pro- 
teid  or  upon  a  diet  containing  absolutely  no  proteid.  The 
ethereal  sulphuric  acid  content  of  the  urine  diminishes  as  the 
total  sulphur  content  diminishes  but  the  percentage  decrease 
is  much  less.  Therefore  when  considered  from  the  stand- 
point of  the  total  sulphuric  acid  content  the  ethereal  sulphuric 
acid  content  is  not  diminished  but  is  increased,  although  the 
total  sulphuric  acid  content  is  diminished.  Folin's  experi- 
ments also  seem  to  show  that  the  indoxyl  sulphuric  acid 
(potassium  indoxyl  sulphate  or  indican)  content  of  the  urine 
does  not  originate  to  any  degree  from  the  metabolism  of 
proteid  material  but  that  it  arises  in  great  part  from  intes- 
tinal putrefaction  and  that  the  excretion  of  indoxyl  sulphuric 
acid  may  alone  be  taken  as  a  rough  index  of  the  extent  of 
putrefactive  processes  within  the  intestine.  Indoxyl  sulphuric 
acid, 

CH 
//\ 

HC        C  — C(0-SO,H), 
I         II       II 

HC        C      CH 

V  \/ 

CH     NH 

therefore,  which  occurs  in  the  urine  as  potassium  indoxyl  sul- 
phate or  indican, 

CH 
//\ 
HC        C  — C(0-SO,K), 

I         II       II 
HC       C      CH 

V  \/ 

CH     NH 

is  clinically  the  most  important  of  the  ethereal  sulphuric  acids. 


URINE. 
Ti  SI  S    FOR    [NDICAN. 

i.  Jaffe's  Test. — Nearly  fill  a  test-tube  with  a  mixture  com- 
posed of  equal  volumes  of  concentrated  HC1  and  the  urine 
under  examination.  Add  2-3  c.c.  of  chloroform  and  a  few 
drops  of  a  calcium  hypochlorite  solution,  place  the  thumb  over 
the  end  of  the  test-tube  and  shake  thoroughly.  The  chloro- 
form is  colored  more  or  less,  according  to  the  amount  of 
indican  present.  Ordinarily  a  blue  color  due  to  the  formation 
of  indigo-blue  is  produced;  less  frequently  a  red  color  due  to 
indigo-red  may  be  noted. 

This  is  the  reaction  (see  also  pages  129  and   130)  : 

CH 

//\ 
HC       C-C-OH 
2     I         ||        II  -'20  = 

HC       C       CH 

V  \/ 

CH    NH 

Indoxyl.  C,H,NO. 

CH  CH 

//\  /\ 

HC       C  —  C  •  0  0  •  C  —  C       CH 

I         I         II  I         II  +  2H20 

HC       C       C  C       C       CH 

V  \/  \/  \// 

CH    NH  XH    CH 

Indigo-blue.  C18H10N502. 

2.  Obermayer's  Test. — Nearly  fill  a  test-tube  with  a  mix- 
ture composed  of  equal  volumes  of  Obermayer's  reagent1  and 
the  urine  under  examination.  Add  2-3  c.c.  of  chloroform, 
place  the  thumb  over  the  end  of  the  test-tube  and  shake  thor- 
oughly.   How  does  this  compare  with  Jaffe's  test? 

C0NHCH2C00H. 

HIPPURIC  ACID,    I 

\/ 

1  Obermayer's  reagent  is  prepared  by  adding  2-4  grams  of  ferric  chlo- 
ride to  a  liter  of  concentrated  HC1   (sp.  gr.   I.19). 


256 


PHYSIOLOGICAL    CHEMISTRY. 


This  acid  occurs  normally  in  the  urine  of  both  the  carnivora 
and  herbivora  but  is  more  abundant  in  the  urine  of  the  latter. 
It  is  formed  by  a  synthesis  of  benzoic  acid  and  glycocoll  which 
takes  place  in  the  kidneys.  The  average  excretion  of  an  adult 
man  for  24  hours  under  normal  conditions  is  about  0.7  gram. 
Hippuric  acid  crystallizes  in  needles  or  rhombic  prisms   (see 

Fig.  92. 


Hippuric   Acid. 

Fig.  92.  above),  the  particular  form  depending  upon  the  ra- 
pidity of  crystallization.  It  is  easily  soluble  in  alcohol  or  hot 
water,  and  only  slightly  soluble  in  ether.  The  output  of  hip- 
puric acid  is  increased  in  diabetes  owing  probably  to  the  inges- 
tion of  much  proteid  and  fruit.  It  is  decreased  in  fevers  and 
in  certain  kidney  disorders  where  the  synthetic  activity  of  the 
renal  cells  is  diminished. 


Experiments  on  Hippuric  Acid. 

1.  Separation  from  the  Urine. — Render  500-1000  c.c.  of 

urine  of  the  horse  or  cow1  alkaline  with  milk  of  lime,  boil  for 

1  If  urine  of  the  horse  or  cow  is  not  available  human  urine  may  serve 
the  purpose  fully  as  well  provided  means  are  taken  to  increase  its  content 
of  hippuric  acid.     This  may  be  conveniently  accomplished  by  ingesting  2 


URINE.  257 

a  few  moments  and  filter  while  hot.  Concentrate  the  filtrate, 
over  a  burner,  to  a  small  volume.  Cool  the  solution,  acidify  it 
strongly  with  concentrated  hydrochloric  acid  and  stand  it  in  a 
cool  place  for  24  hours.  Filter  off  the  crystals  of  hippuric 
acid  which  have  formed  and  wash  them  with  a  little  cold- 
water.  Remove  the  crystals  from  the  paper,  dissolve  them 
in  a  very  small  amount  of  hot  water  and  percolate  the  hot 
solution  through  thoroughly  washed  animal  charcoal,  being 
cue ful  to  wash  out  the  last  portion  of  the  hippuric  acid  solu- 
tion with  hot  water.  Filter,  concentrate  the  filtrate  to  a 
>mall  volume  and  stand  it  aside  for  crystallization.  Examine 
the  crystals  under  the  microscope  and  compare  them  with 
those  in  Fig.  92,  page  256. 

2.  Melting-Point. — Determine  the  melting-point  of  the 
hippuric  acid  prepared  in  the  above  experiment  (see  p.  243). 

3.  Solubility. — Test  the  solubility  of  hippuric  acid  in  the 
ordinary  solvents  (page  4)  and  in  alcohol,  and  ether. 

4.  Formation  of  Nitro-Benzene. — To  a  little  hippuric  acid 
in  a  small  porcelain  dish  add  1-2  c.c.  of  concentrated  HN03 
and  evaporate  to  dryness  on  a  water-bath.  Transfer  the 
residue  to  a  dry  test-tube,  apply  heat  and  note  the  odor  of  the 
artificial  oil  of  bitter  almonds    (nitro-benzene). 

5.  Sublimation. — Place  a  few  crystals  of  hippuric  acid  in 
a  dry  test-tube  and  apply  heat.  The  crystals  are  reduced  to 
an  oily  fluid  which  solidifies  in  a  crystalline  mass  upon  cooling. 
When  stronger  heat  is  applied  the  liquid  assumes  a  red  color 
and  finally  yields  a  sublimate  of  benzoic  acid  and  the  odor  of 
hydrocyanic  acid. 

6.  Formation  of  Ferric  Salt. — Render  a  small  amount  of 
a  solution  of  hippuric  acid  neutral  with  dilute  potassium 
hydroxide.  Now  add  1-3  drops  of  neutral  ferric  chloride 
solution  and  note  the  formation  of  the  ferric  salt  of  hippuric 
acid  as  a  cream  colored  precipitate. 

grams  of  ammonium  benzoate  at  night.     The  fraction  of  urine  passed  in 
the  morning  will  be  found  to  have  a  high  content  of  hippuric  acid.     The 
ammonium  benzoate  is  in  no  way  harmful. 
18 


258  PHYSIOLOGICAL    CHEMISTRY. 

COOH 

OXALIC  ACID,     I 

COOH. 

Oxalic  acid  is  a  constituent  of  normal  urine,  about  0.02 
gram  being  eliminated  in  24  hours.  It  is  present  in  the  urine 
as  calcium  oxalate,  which  is  kept  in  solution  through  the 
medium  of  the  acid  phosphates.  The  origin  of  the  oxalic  acid 
content  of  the  urine  is  not  well  understood.  It  is  eliminated, 
at  least  in  part,  unchanged  when  ingested,  therefore  since 
many  of  the  common  articles  of  diet,  e.  g.,  asparagus,  apples, 
cabbage,  grapes,  lettuce,  spinach,  tomatoes,  etc.,  contain  oxalic 
acid  it  seems  probable  that  the  ingested  food  supplies  a  por- 
tion of  the  oxalic  acid  found  in  the  urine.  There  is  also  ex- 
perimental evidence  that  part  of  the  oxalic  acid  of  the  urine 
is  formed  within  the  organism  in  the  course  of  proteid  and 
fat  metabolism.  It  has  also  been  suggested  that  oxalic  acid 
may  arise  from  an  incomplete  combustion  of  carbohydrates, 
especially  under  certain  abnormal  conditions.  Pathologically, 
oxalic  acid  is  found  to  be  increased  in  amount  in  diabetes 
mellitus,  in  organic  diseases  of  the  liver  and  in  various  other 
conditions  which  are  accompanied  by  a  derangement  of  the 
oxidation  mechanism.  An  abnormal  increase  of  oxalic  acid 
is  termed  oxahtria.  A  considerable  increase  in  the  content  of 
oxalic  acid  may  be  noted  unaccompanied  by  any  other  apparent 
symptom.  Calcium  oxalate  crystallizes  in  at  least  two  distinct 
forms,  dumb-bells  and  octahedra  (Fig.  99,  page  320). 

Experiments. 

1.  Preparation  of  Calcium  Oxalate. — Place  200-250  c.c. 
of  urine  in  a  beaker,  add  10  drops  of  a  saturated  solution  of 
oxalic  acid  and  stand  the  beaker  aside  in  a  cool  place  for  24 
hours.  Examine  the  sediment  under  the  microscope  and  com- 
pare the  crystalline  forms  with  those  shown  in  Fig.  99,  p.  320. 

2.  Solubility. — Test  the  solubility  of  calcium  oxalate  in 
the  ordinary  solvents  (page  4)  and  in  acetic  and  hydrochloric 
acids. 


URINE. 


-59 


NEUTRAL  SULPHUR  COMPOUNDS. 

Under  this  head  may  be  classed  such  bodies  as  cystin  (see 
p.  76),  chondroitin-sulphuric  acid,  oxyproteic  acid,  alloxypro- 
teic  acid,  uroferric  acid,  sulphocyanides  and  taurin  derivatives. 
The  sulphur  content  of  the  bodies  just  enumerated  is  generally 
termed  loosely  combined  or  neutral  sulphur  in  order  thai  it  may 
not  be  confused  with  the  acid  sulphur  which  occurs  in  the  in- 
organic sulphuric  acid  and  ethereal  sulphuric  acid  forms. 
Ordinarily  the  neutral  sulphur  content  of  normal  human  urine 
is  14-20  per  cent  of  the  total  sulphur  content. 

XII-CH-HN 

I  I 

ALLANTOIN,  00  00. 

I  I 

NH-CO       XFL 

Allantoin  has  been  found  in  the  urine  of  suckling-  calves  as 
well  as  in  that  of  the  dog  and  cat.  It  has  also  been  detected  in 
the  urine  of  infants  within  the  first  eight  days  after  birth,  as 
well  as  in  the  urine  of  adults.     It  is  more  abundant  in  the  urine 


Fig.  93. 


Allan  to  in,    from    Cat's   Urine. 
a  and  b,  Forms  in   which   it  crystallized   from   the  urine  ;   c,  re-crystallized 
allantoin.      (Drawn    from    micro-photographs   furnished   by    Prof.    Lafayette    B. 
Mendel  of  Yale  University.) 


260  PHYSIOLOGICAL    CHEMISTRY. 

of  women  during  pregnancy.  Allantoi'n  is  formed  by  the  oxi- 
dation of  uric  acid  and  the  output  is  increased  by  thymus  or 
pancreas  feeding.  When  pure  it  crystallizes  in  prisms  (Fig. 
93,  p.  259)  and  when  impure  in  granules  and  knobs.  Patho- 
logically, it  has  been  found  increased  in  diabetes  insipidus  and 
in  hysteria  with  convulsions  (Pouchet). 

Experiments. 

1.  Separation  from  the  Urine.1 — Mcissner's  Method. — 
Precipitate  the  urine  with  baryta  water.  Neutralize  the  fil- 
trate carefully  with  dilute  sulphuric  acid,  filter  immediately 
and  evaporate  the  filtrate  to  incipient  crystallization.  Com- 
pletely precipitate  this  warm  fluid  with  95  per  cent  alcohol 
(reserve  the  precipitate).  Decant  or  filter  and  precipitate  the 
solution  by  ether.  Combine  the  ether  and  alcohol  precipitates 
and  extract  with  cold  water  or  hot  alcohol ;  allantoi'n  remains 
undissolved.  Bring  the  allantoi'n  into  solution  in  hot  water  and 
recrystallize. 

Allantoi'n  may  be  determined  quantitatively  by  Loewi's 
method.2 

2.  Preparation  from  Uric  Acid. — Dissolve  4  grams  of  uric 
acid  in  100  c.c.  of  water  rendered  alkaline  with  potassium  hy- 
droxide. Cool  and  car ef idly  add  3  grams  of  potassium  per- 
manganate. Filter,  immediately  acidulate  the  filtrate  with 
acetic  acid  and  allow  it  to  stand  in  a  cool  place  over  night. 
Filter  off  the  crystals  and  wash  them  with  water.  Save  the 
wash  water  and  filtrate,  unite  them  and  after  concentrating 
to  a  small  volume,  stand  away  for  crystallization.  Now  com- 
bine all  the  crystals  and  recrystallize  them  from  hot  water. 
Use  these  crystals  in  the  experiments  which  follow. 

3.  Microscopical  Examination. — Examine  the  crystals 
made  in  the  last  experiment  and  compare  them  with  those 
shown  in  Fig.  93,  page  259. 

1  The  urine  of  the  dog  after  thymus,  pancreas  or  uric  acid  feeding  may 
be  employed. 

2  Archiv  fur  Experimented  Pathologie  und   Pharmakologie,   1900,  xliv, 
p.  20. 


URINE.  -'   i 

4.  Solubility. — Test  the  solubility  of  allantoin  in  the  ordi- 
nary solvents  (  page  4 ). 

5.  Reaction. — Dissolve  a  crystal  in  water  and  test  the  re- 
action to  litmus. 

6.  Furfurol  Test. — Place  a  few  crystals  of  allantoin  on  a 
tesl  tablet  or  in  a  porcelain  dish  and  add  1  2  drops  of  a  con- 
centrated aqueous  solution  of  furfurol  and  1-2  drops  of  con- 
centrated hydrochloric  acid.  Observe  the  formation  of  a 
yellow  color  which  turns  to  a  light  purple  if  allowed  to  stand. 
This  tot  is  given  by  urea  hut  not  by  uric  acid. 

7.  Murexid  Test. — Try  this  test  according'  to  the  directions 
given  on  page  ->4<).      Note  that  allantoin   fails  to  respond. 

8.  Reduction  of  Fehling's  Solution. — Make  this  test  in  the 
usual  way  (see  p.  286)  except  that  the  boiling  must  he  pro- 
longed and  excessive.  I'ltimately  the  allantoin  will  reduce 
the  solution.     Compare  with  the  result  on  uric  acid,  page  J4<>. 

AROMATIC  OXYACIDS. 

Two  of  the  most  important  of  the  oxyacids  are  paraoxy- 
phciiyl-acetic  acid, 

CH2-COOH, 


OH 

and  paraoxyplicnyl-propioiiic  acid, 


CHo-CH.-COOH. 
/V 


OH 

They  are  products  of  the  putrefaction  of  proteid  material  and 
tyrosin  is  an  intermediate  stage  in  their  formation.  Both 
these  acids  for  the  most  part  pass  unchanged  into  the  urine 
where  they  occur  normally  in  very  small  amount.  The  con- 
tent may  be  increased  in  the  same  manner  as  the  phenol  con- 


262  PHYSIOLOGICAL    CHEMISTRY. 

tent,  in  particular  by  acute  phosphorus  poisoning.  A  fraction 
of  the  total  aromatic  oxyacid  content  of  the  urine  is  in  com- 
bination with  sulphuric  acid,  but  the  greater  part  is  present  in 
the  form  of  salts  of  sodium  and  potassium. 

Homogentisic  Acid  or  di-oxyphenyl-acetic  acid. 

OH 


j^CHo-COOH, 


OH 

is  another  important  oxyacid  sometimes  present  in  the  urine. 
Under  the  name  glycosuric  acid  it  was  first  isolated  from  the 
urine  by  Prof.  John  Marshall  of  the  University  of  Pennsylva- 
nia; subsequently  Baumann  isolated  it  and  determined  its 
chemical  constitution.  It  occurs  in  cases  of  alkaptonuria.  A 
urine  containing  this  oxyacid  turns  greenish-brown  from  the 
surface  downward  when  treated  with  a  little  sodium  hydroxide 
or  ammonia.  If  the  solution  be  stirred  the  color  very  soon 
becomes  dark  brown  or  even  black.  Homogentisic  acid  re- 
duces alkaline  copper  solutions  but  not  alkaline  bismuth  solu- 
tions. Uroleucic  acid  is  similar  in  its  reactions  to  homogentisic 
acid. 

Oxymandelic  Acid  or  paraoxyphenyl-glycolic  acid, 


( 


OH 
\ 


CH(OH)-COOH, 

has  been  detected  in  the  urine  in  cases  of  yellow  atrophy  of 
the  liver. 

Kynurenic  Acid  or  y-oxy-/?-quinoline  carbonic  acid, 

CH      COH 

/\  /  V 

HC         C        C-COOH, 

I  II  I 

HC         C        CH 

\/  \/ 

CH      N 


URINE.  263 

is. present  in  the  urine  of  the  dog"  and  has  recently  been  detected 
by  Swain  in  the  urine  of  the  coyote.  To  isolate  it  from  the 
urine  proceed  as  follows:  Acidify  the  urine  with  hydrochloric 
acid  in  the  proportion  1  125.  From  this  acid  fluid  both  the  uric 
acid  and  the  kynurenic  acid  separate  in  the  course  of  24-48 
hours.  Filter  off  the  combined  crystalline  deposit  of  the  two 
acids,  dissolve  the  kynurenic  acid  in  dilute  ammonia  (uric 
acid  is  insoluble)  and  reprecipitate  it  with  hydrochloric  acid. 
Kynurenic  acid  may  be  quantitatively  determined  by  Capal- 
di's  method.1 

COOII. 


BENZOIC  ACID,  | 

V 

Benzoic  acid  has  been  detected  in  the  urine  of  the  rabbit  and 
dog.  It  is  also  said  to  occur  in  human  urine  accompanying 
renal  disorders.  The  benzoic  acid  probably  originates  from  a 
fermentative  decomposition  of  the  hippuric  acid  of  the  urine. 

Experiments. 

1.  Solubility. — Test  the  solubility  of  benzoic  acid  in  water, 
alcohol  and  ether. 

2.  Crystalline  Form. — Recrystallize  some  benzoic  acid  from 
hot  water,  examine  the  crystals  under  the  microscope  and  com- 
pare them  with  those  reproduced  in  Fig.  94,  page  264. 

3.  Sublimation. — Place  a  little  benzoic  acid  in  a  test-tube 
and  heat  over  a  flame.  Note  the  odor  which  is  evolved  and 
observe  that  the  acid  sublimes  in  the  form  of  needles. 

4.  Dissolve  a  little  sodium  benzoate  in  water  and  add  a  solu- 
tion of  neutral  ferric  chloride.  Note  the  production  of  a 
brownish-yellow  precipitate  (Salicylic  acid  gives  a  reddish- 
violet  color  under  the  same  conditions).  Add  ammonium  hy- 
droxide to  some  of  the  precipitate.  It  dissolves  and  ferric  hy- 
droxide is  formed.  Add  a  little  hydrochloric  acid  to  another 
portion  of  the  original  precipitate  and  stand  the  vessel  away 
over  night.     What  do  you  observe? 

1  Zeitschrift  fur  physiologische  Chemie,  1897,  xxiii,  p.  92. 


264 


PHYSIOLOGICAL    CHEMISTRY, 
Fig.  94. 


Benzoic    Acid. 


NUCLEO-PROTEID. 

The  nubecula  of  normal  urine  has  been  shown  by  one  investi- 
gator to  consist  of  a  mucoid  containing  12.7  per  cent  of  nitro- 
gen and  2.3  per  cent  of  sulphur.  This  body  evidently  origi- 
nates in  the  urinary  passages.  It  is  probably  slightly  soluble 
in  the  urine.  Some  investigators  believe  that  the  body  form- 
ing the  nubecula  of  normal  urine  is  nucleo-proteid  and  not  a 
mucin  or  mucoid  as  stated  above.  A  discussion  of  nucleo- 
proteid  and  related  bodies  occurring  in  the  urine  under  patho- 
logical conditions  will  be  found  on  page  296. 

NH-CO 
I 

OXALURIC  ACID,  CO 

NH2  COOH. 

Oxaluric  acid  is  not  a  constant  constituent  of  normal  human 
urine,  and  when  found  occurs  only  in  traces  as  the  ammonium 
salt.  Upon  boiling  oxaluric  acid  it  splits  into  oxalic  acid  and 
urea. 


URINE.  265 

ENZYMES. 

Various  types  of  enzymes  have  been  isolated  from  the  urine. 
Pepsin,  which  probably  originates  in  the  stomach,  and  a 
diastatic  enzyme  have  been  more  carefully  studied  than  the 
•  •ther  forms.  The  presence  of  trypsin  and  rennin  in  the  urine 
is  questioned. 

VOLATILE   FATTY  ACIDS. 

Acetic,  butyric  and  formic  acids  have  been  found  under 
normal  conditions  in  the  urine  of  man  and  of  certain  carnivora 
as  well  as  in  the  urine  of  herbivora.  Normally  they  arise  prin- 
cipally from  the  fermentation  of  carbohydrates  and  the  putre- 
faction of  proteids.  The  acids  containing  the  fewest  carbon 
atoms  (formic  and  acetic)  are  found  to  be  present  in  larger 
percentage  than  those  which  contain  a  larger  number  of  such 
atoms.  The  volatile  fatty  acids  occur  in  normal  urine  in 
traces,  the  total  output  for  twenty- four  hours,  according  to 
different  investigators,  varying  from  0.008  gram  to  0.05  gram. 

Pathologically,  the  excretion  of  volatile  fatty  acids  is  in- 
creased in  diabetes,  fevers,  and  in  certain  hepatic  diseases  in 
which  the  parenchyma  of  the  liver  is  seriously  affected.  Under 
other  pathological  conditions  the  output  may  be  diminished. 
These  variations,  however,  in  the  excretion  of  the  volatile 
fatty  acids  possess  very  little  diagnostic  value. 

CH3 

PARALACTIC  ACID,   CH(OH) 

COOH. 

Paralactic  acid  is  supposed  to  pass  into  the  urine  when  the 
supply  of  oxygen  in  the  organism  is  diminished  through  any 
cause,  e.  g.,  after  acute  yellow  atrophy  of  the  liver,  acute 
phosphorus  poisoning  or  epileptic  attacks.  This  acid  has  also 
been  found  in  the  urine  of  healthy  persons  following  the 
physical  exercise  incident  to  prolonged  marching.     Paralactic 


266  PHYSIOLOGICAL    CHEMISTRY. 

acid  has  been  detected  in  the  urine  of  birds  after  the  removal 
of  the  liver. 

CH„  •  CO  •  NH  •  CHo  •  COOH. 
/\ 

PHENACETURIC  ACID, 


Phenaceturic  acid  occurs  principally  in  the  urine  of  herbivor- 
ous animals  but  has  frequently  been  detected  in  human  urine. 
It  is  produced  in  the  organism  through  the  synthesis  of  glyco- 
coll  and  phenylacetic  acid.  It  may  be  decomposed  into  its 
component  parts  by  boiling  with  dilute  mineral  acids.  The 
crystalline  form  of  phenaceturic  acid  (small  rhombic  plates 
with  rounded  angles)  resembles  one  form  of  uric  acid  crystal. 

PHOSPHORIZED  COMPOUNDS. 

Phosphorus  in  organic  combination  has  been  found  in  the 
urine  in  such  bodies  as  glycerophosphoric  acid,  which  may 
arise  from  the  decomposition  of  lecithin,  and  phosphocarnic 
acid.  It  is  claimed  that  on  the  average  about  2.5  per  cent  of 
the  total  phosphorus  elimination  is  in  organic  combination. 

PIGMENTS. 

There  are  at  least  three  pigments  normally  present  in  human 
urine.  These  pigments  are  uro chrome,  urobilin  and  uroery- 
thrin. 

A.    UROCHROME. 

This  is  the  principal  pigment  of  normal  urine  and  imparts  the 
characteristic  yellow  color  to  that  fluid.  It  is  apparently  closely 
related  to  its  associated  pigment  urobilin  since  the  latter  may  be 
readily  converted  into  urochrome  through  evaporation  of  its 
aqueous-ether  solution.  Urochrome  may  be  obtained  in  the 
form  of  a  brown,  amorphous  powder  which  is  readily  soluble 
in  water  and  95  per  cent  alcohol.  It  is  less  soluble  in  absolute 
alcohol,  acetone,  amyl  alcohol  and  acetic  ether  and  insoluble  in 
benzene,  chloroform  and  ether.  Urochrome  is  said  to  be  a 
nitrogenous  body  (4.2  per  cent  nitrogen),  free  from  iron. 


URINE.  267 

B.    UROBILIN. 

Urobilin,  which  was  at  one  time  considered  to  be  the  princi- 
pal pigment  of  urine,  in  reality  contributes  little  toward  the 
pigmentation  of  this  fluid.  It  is  claimed  thai  no  urobilin  is 
present  in  freshly  voided  normal  urine  but  that  its  precursor,  a 
chromogen  called  urobilinogen,  is  presenl  and  gives  rise  to 
ur<  bilin  upon  decomposition  through  the  influence  of  light.  It 
is  claimed  by  some  investigators  that  there  are  various  forms 
oi  urobilin,  e.  g.}  normal,  febrile,  physiological  and  patholog- 
ical. Urobilin  is  said  to  be  very  similar  to.  if  not  absolutely 
identical  with,  hydrobilirubin  (see  page  140). 

Urobilin  may  be  obtained  as  an  amorphous  powder  which 
varies  in  color  from  brown  to  reddish-brown,  red  and  reddish- 
yellow  depending  upon  the  way  in  which  it  is  prepared.  It  is 
easily  soluble  in  ethyl  alcohol,  amyl  alcohol  and  chloroform, 
and  slightly  soluble  in  ether,  acetic  ether  and  in  water.  Its 
solutions  show  characteristic  absorption-bands  (see  Absorp- 
tion Spectra,  Plate  II).  Under  normal  conditions  urobilin 
is  derived  from  tbe  bile  pigments  in  the  intestine. 

Urobilin  is  increased  in  most  acute  infectious  diseases  such 
as  erysipelas,  malaria,  pneumonia  and  scarlet  fever.  It  is  also 
increased  in  appendicitis,  carcinoma  of  the  liver,  catarrhal 
icterus,  pernicious  aiucmia  and  in  cases  of  poisoning  by  anti- 
febrin,  antipyrin,  pyridin.  and  potassium  chlorate.  In  gen- 
eral it  is  usually  increased  when  blood  destruction  is  excessive 
and  in  many  disturbances  of  the  liver.  It  is  markedly  de- 
creased in  phosphorus  poisoning. 

Experiments. 

1.  Spectroscopic  Examination. — Acidify  the  urine  with 
HC1  and  allow  it  to  remain  exposed  to  the  air  for  a  few 
moments.  By  this  means  if  any  urobilinogen  is  present  it 
will  be  transformed  into  urobilin.  The  urine  may  now  be 
examined  by  means  of  the  spectroscope.  If  urobilin  is  present 
in  the  fluid  the  characteristic  absorption-band  lying  between  b 
and  F  will  be  observed  (see  Absorption  Spectra,  Plate  II).     It 


268 


PHYSIOLOGICAL    CHEMISTRY 


may  be  found  necessary  to  dilute  the  urine  with  water  before 
a  distinct  absorption-band  is  observed.  This  test  may  be'modi- 
fied  by  acidifying  10  c.c.  of  urine  with  HC1  and  shaking  it 
gently  with  5  c.c.  of  amyl  alcohol.  The  alcoholic  extract  when 
examined  spectroscopically  will  show  the  characteristic  urobilin 
absorption-band.  (Note  the  spectroscopic  examination  in  the 
next  experiment.) 

2.  Ammoniacal-Zinc  Chloride  Test. — Render  some  of  the 
urine  ammoniacal  by  the  addition  of  ammonium  hydroxide, 
and  after  allowing  it  to  stand  a  short  time  filter  off  the  preci- 
tate  of  phosphates  and  add  a  few  drops  of  zinc  chloride 
solution  to  the  filtrate.  Observe  the  production  of  a  greenish 
fluorescence.  Examine  the  fluid  by  means  of  the  spectroscope 
and  note  the  absorption-band  which  occupies  much  the  same 
position  as  the  absorption-band  of  urobilin  in  acid  solution 
(see  Absorption  Spectra,  Plate  II). 

3.  Gerhardt's  Test. — To  20  c.c.  of  urine  add  3-5  c.c.  of 
chloroform  and  shake  well.  Separate  the  chloroform  extract 
and  add  to  it  a  few  drops  of  iodine  solution  (I  in  KI) .  Render 
the  mixture  alkaline  with  a  dilute  solution  of  potassium  hy- 
droxide and  note  the  production  of  a  yellow  or  yellowish-brown 
color.  The  solution  ordinarily  exhibits  a  greenish  fluores- 
cence. 

4.  Wirsing's  Test. — To  20  c.c.  of  urine  add  3-5  c.c.  of 
chloroform  and  shake  gently.  Separate  the  chloroform  ex- 
tract and  add  to  it  a  drop  of  an  alcoholic  solution  of  zinc 
chloride.  Note  the  rose-red  color  and  the  greenish  fluores- 
cence. If  the  solution  is  turbid  it  may  be  rendered  clear  by 
the  addition  of  a  few  c.c.  of  absolute  alcohol. 

5.  Ether- Absolute  Alcohol  Test. — Mix  urine  and  pure 
ether  in  equal  volumes  and  shake  gently  in  a  separatory  funnel. 
Separate  the  ether  extract,  evaporate  it  to  dryness  and  dissolve 
the  residue  in  2-3  c.c.  of  absolute  alcohol.  Note  the  greenish 
fluorescence.  Examine  the  solution  spectroscopically  and 
observe  the  characteristic  absorption-band  (see  Absorption 
Spectra,  Plate  II). 


URI  X  l  269 

6.  Ring  Test. — Acidify  25  c.c.  of  urine  with  -'  3  drops  of 
concentrated  HC1,  add  5  c.c.  of  chloroform  and  shake  the 
mixture.  Separate  the  chloroform,  place  it  in  a  test-tube  and 
add  carefully  3  5  c.c.  of  an  alcoholic  solution  of  zinc  acetate. 
Observe  the  formation  of  a  green  ring  at  the  zone  of  contact 
of  the  two  fluids.  If  the  tube  is  shaken  a  fluorescence  may  he 
observed. 

C.    UROERYTHRIN. 

This  pigment  is  Frequently  present  in  small  amount  in  nor- 
mal urine.  The  red  color  of  urinary  sediments  is  due  in  great 
part  to  the  presence  of  uroerythrin.  It  is  easily  soluble  in  amy! 
alcohol,  slightly  soluble  in  acetic  ether,  absolute  alcohol  or 
chloroform,  and  nearly  insoluble  in  water.  Dilute  solutions 
of  uroerythrin  are  pink  in  color  while  concentrated  solutions 
are  orange-red  or  bright  red :  none  of  its  solutions  fluoresce. 
Uroerythrin  is  increased  in  amount  after  strenuous  physical 
exercise,  digestive  disturbances,  fevers,  certain  liver  disorders 
and  in  various  other  pathological  conditions. 

PTOMAINES   AND    LEUCOMAINES. 

These  toxic  substances  are  said  to  be  present  in  small  amount 
in  normal  urine.  It  is  claimed  that  five  different  poisons  may 
be  detected  in  the  urine,  and  it  is  further  stated  that  each  of 
these  substances  produces  a  specific  and  definite  symptom  when 
injected  intravenously  into  a  rabbit.  The  resulting  symptoms 
are  narcosis,  salivation,  mydriasis,  paralysis  and  convulsions 
The  day  urine  is  principally  narcotic  and  is  2-4  times  as  toxic 
as  the  night  urine  which  is  chiefly  productive  of  convulsions. 

PURIN   BASES. 

The  purin  bases  found  in  human  urine  are  adenin.  carnin, 
epiguanin,  episarkin,  guanin,  xanthin,  heteroxanthin,  hypo- 
xanthin,  paraxanthin  and  i-methylxanthin.  The  main  bulk  of 
the  purin  base  content  of  the  urine  is  made  up  of  paraxanthin, 
heteroxanthin  and  i-methvlxanthin  which  are  derived  for  the 


270  PHYSIOLOGICAL    CHEMISTRY. 

most  part  from  the  caffein,  theobromin  and  theophyllin  of  the 
food.  The  total  purin  base  content  is  made  np  of  the  products 
of  two  distinct  forms  of  metabolism,  i.  e.,  metabolism  of  in- 
gested nucleins  and  purins  and  metabolism  of  tissue  nuclein 
material.  Purin  bases  resulting  from  the  first  form  of  meta- 
bolism are  said  to  be  of  exogenous  origin  whereas  those  re- 
sulting from  the  second  form  of  metabolism  are  said  to  be  of 
endogenous  origin.  The  daily  output  of  purin  bases  by  the 
urine  is  extremely  small  and  varies  greatly  with  the  individual 
(16-60  milligrams).  The  output  is  increased  after  the  inges- 
tion of  nuclein  material  as  well  as  after  the  increased  destruc- 
tion of  leucocytes.  A  well  marked  increase  accompanies  leu- 
kaemia. Edsall  has  very  recently  shown  that  the  output  of 
purin  bases  by  the  urine  is  increased  as  a  result  of  X-ray  treat- 
ment. 

Experiment. 

1.  Formation  of  the  Silver  Salts. — Add  an  excess  of  mag- 
nesia mixture1  to  25  c.c.  of  urine.  Filter  off  the  precipitate 
and  add  ammoniacal  silver  solution2  to  the  filtrate.  A  precipi- 
tate composed  of  the  silver  salts  of  the  various  purin  bases  is 
produced. 

2.  Inorganic  Physiological  Constituents. 
Ammonia. 

Next  to  urea,  ammonia  is  the  most  important  of  the  nitro- 
genous end-products  of  proteid  metabolism.  Ordinarily  about 
4.6-5.6  per  cent  of  the  total  nitrogen  of  the  urine  is  eliminated 
as  ammonia  and  on  the  average  this  would  be  about  0.7  gram 
per  day.  Under  normal  conditions  the  ammonia  is  present  in 
the  urine  in  the  form  of  the  chloride,  phosphate  or  sulphate. 
This  is  due  to  the  fact  that  combinations  of  this  sort  are  not 

1  Magnesia  mixture  may  be  prepared  as  follows :  Dissolve  175  grams  of 
MgSO.,  and  350  grams  of  NH4C1  in  1400  c.c.  of  distilled  water.  Add  700 
grams  of  concentrated  NEUOH,  mix  very  thoroughly  and  preserve  the 
mixture  in  a  glass-stoppered  bottle. 

2  Ammoniacal  silver  solution  may  be  prepared  according  to  directions 
given   on  page  377. 


URINE.  -71 

oxidized  in  the  organism  to  form  urea,  1  >n t  arc  excreted  as  such. 

This  explains  the  increase  in  the  OUtpUl  of  ammonia  which  fol- 
lows the  administration  of  the  ammonium  salts  of  the  mineral 
acids  or  of  the  acids  themselves.  On  the  other  hand  when 
ammonium  acetate  and  many  other  ammonium  salts  of  certain 
organic  acids  are  administered  no  increase  in  the  output  of 
ammonia  occurs  since  the  salt  is  oxidized  and  its  nitrogen  ulti- 
mately appears  in  the  urine  as  urea. 

The  acids  formed  during  the  process  of  proteid  destruction 
within  the  body  have  an  influence  upon  the  excretion  of  am- 
monia similar  to  that  exerted  by  acids  which  have  been  admin- 
istered. Therefore  a  pathological  increase  in  the  output  of 
ammonia  is  observed  in  such  diseases  as  are  accompanied  by 
an  increased  and  imperfect  proteid  metabolism,  and  especially 
in  diabetes,  in  which  disease  diacetic  acid  and  /8-oxybutyric 
acid  are  found  in  the  urine  in  combination  with  the  ammonia. 

As  the  result  of  recent  experiments  Folin  claims  that  a 
pronounced  decrease  in  the  extent  of  proteid  metabolism, 
as  measured  by  the  total  nitrogen  in  the  urine,  is  frequently 
accompanied  by  a  decreased  elimination  of  ammonia.  The 
ammonia  elimination  is  therefore  probably  determined  by 
other  factors  than  the  total  proteid  catabolism  as  such.  Fur- 
thermore, he  believes  that  a  decided  decrease  in  the  total 
nitrogen  excretion  is  always  accompanied  by  a  relative  increase 
in  the  ammonia-nitrogen,  provided  the  food  is  of  a  character 
yielding  an  alkaline  ash. 

The  quantitative  determination  of  ammonia  must  be  made 
upon  the  fresh  urine  since  upon  standing  the  normal  urine  will 
undergo  ammoniacal  fermentation    (see  page  230). 

Sulphates. 

Sulphur  in  combination,  is  excreted  in  two  forms  in  the 
urine;  first,  as  loosely  combined,  unoxidized  or  neutral  sulphur 
and  second,  as  oxidized  or  acid  sulphur.  The  loosely  combined 
sulphur  is  excreted  mainly  as  a  constituent  of  such  bodies  as 
cystin,  cystein,  taurin,  hydrogen  sulphide,  ethyl  sulphide,  sul- 


272  PHYSIOLOGICAL    CHEMISTRY. 

phocyanides,  sulphonic  acids,  oxyproteic  acid,  alloxyproteic  acid 
and  uroferric  acid.  The  amount  of  loosely  combined  sulphur 
eliminated  is  in  great  measure  independent  of  the  extent  of  pro- 
teid  decomposition  or  of  the  total  sulphur  excretion.  In  this 
characteristic  it  is  somewhat  similar  to  the  excretion  of  cre- 
atinin.  The  oxidised  sulphur  is  eliminated  in  the  form  of  sul- 
phuric acid,  principally  as  salts  of  sodium,  potassium,  calcium 
and  magnesium ;  a  relatively  small  amount  occurs  in  the  form 
of  ethereal  sulphuric  acid,  i.  c,  sulphuric  acid  in  combination 
with  such  aromatic  bodies  as  phenol,  indol,  skatol,  cresol,  pyro- 
catechin  and  hydroquinone.  Sulphuric  acid  in  combination 
with  Na,  K,  Ca  or  Mg  is  sometimes  termed  inorganic  or  pre- 
formed sulphuric  acid  whereas  the  ethereal  sulphuric  acid  is 
sometimes  called  conjugate  sulphuric  acid.  The  greater  part 
of  the  sulphur  is  eliminated  in  the  oxidized  form  but  the  abso- 
lute percentage  of  sulphur  excreted  as  the  preformed,  ethereal 
or  loosely  combined  type  depends  upon  the  total  quantity  of 
sulphur  present,  i.  e.,  there  is  no  definite  ratio  between  the 
three  forms  of  sulphur  which  will  apply  under  all  conditions. 
The  preformed  sulphuric  acid  may  be  precipitated  directly  from 
acidified  urine  with  BaCl2,  whereas  the  ethereal  sulphuric  acid 
must  undergo  a  preliminary  boiling  in  the  presence  of  a  mineral 
acid  before  it  can  be  so  precipitated. 

The  sulphuric  acid  excreted  by  the  urine  arises  principally 
from  the  oxidation  of  proteid  material  within  the  body;  a 
relatively  small  amount  is  due  to  ingested  sulphates.  Under 
normal  conditions  about  2.5  grams  of  sulphuric  acid  is  elimi- 
nated daily.  Since  the  sulphuric  acid  content  of  the  urine  has, 
for  the  most  part,  a  proteid  origin  and  since  one  of  the  most 
important  constituents  of  the  proteid  molecule  is  nitrogen,  it 
would  be  reasonable  to  suppose  that  a  fairly  definite  ratio  might 
exist  between  the  excretion  of  these  two  elements.  However, 
when  we  appreciate  that  the  percentage  content  of  N  and  S 
present  in  different  proteids  is  subject  to  rather  wide  variations, 
the  fixing  of  a  ratio  which  will  express  the  exact  relation 
existing  between  these  two  substances,  as  they  appear  in  the 


URINE.  273 

urine  as  end-products  of  proteid  metabolism,  is  practically 
impossible.  It  \ya<  lieen  suggested  that  the  ratio  5  :i  expresses 
this  relation  in  a  general  way. 

Pathologically,  the  excretion  of  sulphuric  acid  by  the  urine 
is  increased  in  acute  fevers  and  in  all  other  diseases  marked  by 
a  stimulated  metabolism,  whereas  a  decrease  in  the  sulphuric 
acid  excretion  is  observed  in  those  diseases  which  are  accom- 
panied by  a  loss  of  appetite  and  a  diminished  metabolic  activity. 

Experiments. 

1.  Detection  of  Inorganic  Sulphuric  Acid. — Place  about 
10  c.c.  of  urine  in  a  test-tube,  acidify  with  acetic  acid  and  add 
some  barium  chloride  solution.  A  white  precipitate  of  barium 
sulphate  forms. 

2.  Detection  of  Ethereal  Sulphuric  Acid. — Filter  off  the 
barium  sulphate  precipitate  formed  in  the  above  experiment, 
add  1  c.c.  of  hydrochloric  acid  and  a  little  barium  chloride  solu- 
tion to  the  filtrate  and  heat  the  mixture  to  boiling  for  1-2 
minutes.  Xote  the  appearance  of  a  turbidity  due  to  the  pres- 
ence of  sulphuric  acid  which  has  been  separated  from  the 
ethereal  sulphates  and  has  combined  with  the  barium  of  the 
BaCU  to  form  BaSO,. 

3.  Detection  of  Loosely  Combined  or  Neutral  Sulphur. 
— Place  about  10  c.c.  of  urine  in  a  test-tube,  introduce  a  small 
piece  of  zinc,  add  sufficient  hydrochloric  acid  to  cause  a  gentle 
evolution  of  hydrogen  and  over  the  mouth  of  the  tube  place 
a  filter  paper  saturated  with  plumbic  acetate  solution.  In  a 
short  time  the  portion  of  the  paper  in  contact  with  the  vapors 
within  the  test-tube  becomes  blackened  due  to  the  formation 
of  lead  sulphide.  The  nascent  hydrogen  has  reacted  with  the 
loosely  combined  or  neutral  sulphur  to  form  hydrogen  sulphide 
and  this  gas  coming  in  contact  with  the  plumbic  acetate  paper 
has  caused  the  production  of  the  black  lead  sulphide.  Sul- 
phur in  the  form  of  inorganic  or  ethereal  sulphuric  acid  does 
not  respond  to  this  test. 

4.  Calcium  Sulphate  Crystals. — Place  10  c.c.  of  urine  in  a 
19 


274 


PHYSIOLOGICAL    CHEMISTRY. 


test-tube,  add  10  drops  of  calcium  chloride  solution  and  allow 
the  tube  to  stand  until  crystals  form.  Examine  the  calcium 
sulphate  crystals  under  the  microscope  and  compare  them  with 

those  shown  in  Fig.  95,  p.  274. 


Fig.  95. 


Chlorides. 


Calcium     Sulphate. 
and    Weil.) 


(Hensel 


Next  to  urea,  the  chlorides 
constitute  the  chief  solid  con- 
stituent of  the  urine.  The  prin- 
cipal chlorides  found  in  the 
urine  are  those  of  sodium,  po- 
tassium, ammonium  and  mag- 
nesium, with  sodium  chloride 
predominating".  The  excretion 
of  chlorides  is  dependent,  in 
great  part,  upon  the  nature  of  the  diet,  but  on  the  average  the 
daily  output  is  about  10-15  grams,  expressed  as  sodium  chlo- 
ride. Copious  water-drinking  increases  the  output  of  chlorides 
considerably.  Because  of  their  solubility,  chlorides  are  never 
found  in  the  urinary  sediment. 

Since  the  amount  of  chlorides  excreted  in.  the  urine  is  due 
primarily  to  the  chloride  content  of  the  food  ingested,  it  fol- 
lows that  a  decrease  in  the  amount  of  ingested  chloride  will 
likewise  cause  a  decrease  in  the  chloride  content  of  the  urine 
In  cases  of  actual  fasting  the  chloride  content  of  the  urine  may 
be  decreased  to  a  slight  trace  which  is  derived  from  the  body 
fluids  and  tissues.  Under  these  conditions,  however,  an  exam- 
ination of  the  blood  of  the  fasting  subject  will  show  the  per- 
centage of  chlorides  in  this  fluid  to  be  approximately  normal. 
This  forms  a  very  striking  example  of  the  care  nature  takes 
to  maintain  the  normal  composition  of  the  blood.  There  is  a 
limit  to  the  power  of  the  body  to  maintain  this  equilibrium, 
however,  and  if  the  fasting  organism  be  subjected  to  the  influ- 
ence of  diuretics  for  a  time,  a  point  is  reached  where  the  com- 
position of  the  blood  can  no  longer  be  maintained  and  a  gradual 
decrease  in  its  chloride  content  occurs  which  finally  results  in 


URINE.  275 

death.  Death  is  supposed  to  result  not  so  much  because  of  a 
lack  of  chlorine  as  from  a  deficiency  of  sodium.  This  is  shown 
from  the  fact  that  potassium  chloride,  for  instance,  cannot  re- 
place the  sodium  chloride  of  the  blood  when  the  latter  is  de- 
creased in  the  manner  above  slated.  When  this  substitution 
is  attempted  the  potassium  salt  is  excreted  at  once  in  the  urine, 
and  death  follows  as  above  indicated. 

Pathologically,  the  excretion  of  chlorides  may  be  decreased 
in  some  fevers,  chronic  nephritis,  croupous  pneumonia,  diar- 
rhoea, certain  stomach  disorders  and  in  acute  articular  rheu- 
matism. 

Experiment. 

Detection  of  Chlorides  in  Urine. — Place  about  5  c.c.  of 
urine  in  a  test-tube,  render  it  acid  with  nitric  acid  and  add  a 
few  drops  of  a  solution  of  argentic  nitrate.  A  white  precipi- 
tate, due  to  the  formation  of  argentic  chloride,  is  produced. 
This  precipitate  is  soluble  in  ammonium  hydroxide. 

Phosphates. 

Phosphoric  acid  exists  in  the  urine  in  two  general  forms: 
First,  that  in  combination  with  the  alkali  metals,  sodium  and 
potassium,  and  the  radical  ammonium;  second,  that  in  combi- 
nation with  the  alkaline  earths,  calcium  and  magnesium.  Phos- 
phates formed  through  a  union  of  phosphoric  acid  with  the 
alkali  metals  are  termed  alkaline  phosphates,  or  phosphates  of 
the  alkali  metals,  whereas  phosphates  formed  through  a  union 
of  phosphoric  acid  with  the  alkaline  earths  are  termed 
earthy  phosphates,  or  phosphates  of  the  alkaline  earths. 

Three  series  of  salts  are  formed  by  phosphoric  acid : 
Normal,  M...PO4,1  mono-hydrogen,  M2HP04,  and  di-hydro- 
gen,  MH2P04.  The  di-hydrogen  salts  are  acid  in  reaction 
and  it  was  generally  believed  that  about  60  per  cent  of  the 
total  phosphate  content  of  the  urine  was  in  the  form  of  this 
type  of  salt,  and  that  the  acidity  of  the  urine  was  due  in  great 
part  to  the  presence  of  sodium  di-hydrogen  phosphate,     Re- 

1  M  may  be  occupied  by  any  of  the  alkali  metals  or  alkaline  earths. 


276  PHYSIOLOGICAL    CHEMISTRY. 

cently,  however,  it  has  been  quite  clearly  shown  that  the  nor- 
mal acidity  of  the  urine  is  not  due  to  the  presence  of  this 
salt  but  is  due.  at  least  in  part,  to  the  presence  of  various 
acidic  radicals.  In  this  connection  Folin  believes  that  the 
phosphates  in  clear  acid  urine  are  all  of  the  mono-hydrogen 
type,  and  that  the  acidity  of  the  urines  of  this  character  is 
generally  greater  than  the  combined  acidity  of  all  the  phos- 
phates present;  the  excess  in  the  acidity  above  that  due  to 
phosphates  he  believes  to  be  due  to  free  organic  acids.  In 
bones  the  phosphates  occur  principally  in  the  form  of  the 
normal  salts  of  calcium  and  magnesium.  The  mono-hydro- 
gen salts  as  a  class  are  alkaline  in  reaction  to  litmus,  and  it  is 
to  the  presence  of  di-sodium  hydrogen  phosphate,  Na2HP04, 
that  the  greater  part  of  the  alkalinity  of  the  saliva  is  due. 

The  excretion  of  phosphoric  acid  is  extremely  variable  but 
on  the  average  the  total  output  for  24  hours  is  about  2.5 
grams,  expressed  as  P205.  Ordinarily  the  total  output  is 
distributed  between  alkaline  phosphates  and  earthy  phos- 
phates approximately  in  the  ratio  2:1.  The  greater  part 
of  this  phosphoric  acid  arises  from  the  ingested  food,  either 
from  the  preformed  phosphates  or  more  especially  from 
the  phosphorus  in  organic  combination  such  as  we  find  it 
in  phospho-proteids,  nucleo-proteids,  nncleins  and  lecithins; 
the  phosphorus-containing  tissues  of  the  body  also  contrib- 
ute to  the  total  output  of  this  element.  Alkaline  phos- 
phates ingested  with  the  food  have  a  tendency  to  increase 
the  phosphoric  acid  content  of  the  urine  to  a  greater  extent 
than  the  earthy  phosphates  so  ingested.  This  is  due,  in  a 
measure,  to  the  fact  that  a  portion  of  the  earthy  phosphates, 
under  certain  conditions,  may  be  precipitated  in  the  intes- 
tine and  excreted  in  the  feces;  this  is  especially  to  be  noted  in 
the  case  of  herbivorous  animals.  Since  the  extent  to  which 
the  phosphates  are  absorbed  in  the  intestine  depends  upon 
the  form  in  which  they  are  present  in  the  food,  under  ordi- 
nary conditions,  there  can  be  no  absolute  relationship  be- 
tween the  urinary  output  of   nitrogen  and  phosphorus.     If 


URINE.  277 

the  diel  is  constant,  however,  from  day  to  day,  thus  allowing 
of  the  preparation  of  both  a  nitrogen  and  a  phosphorus  bal- 
ance,1 a  definite  ratio  may  be  established.  In  experiments 
upon  dog-,,  which  were  fed  an  exclusive  meat  diet,  the  ratio 
of  nitrogen  to  phosphorus,  in  the  urine  ami  free-,  was  found 
to  be  8. 1  :  1 . 

Pathologically  the  excretion  of  phosphoric  acid  is  increased 
in  such  diseases  of  the  hones  as  diffuse  periostosis,  osteoma- 
lacia and  rickets:  according  to  some  investigators,  in  the  early 
stages  of  pulmonary  tuberculosis;  in  acute  yellow  atrophy  of 
the  liver;  in  diseases  which  are  accompanied  by  an  extensive 
decomposition  of  nervous  tissue  and  after  sleep  induced  by 
potassium  bromide  or  chloral  hydrate  (Mendel).  It  is  also 
increased  after  copious  water-drinking.  A  decrease  in  the 
excretion  of  phosphates  is  at  times  noted  in  febrile  affections, 
such  as  the  acute  infectious  diseases;  in  pregnancy,  in  the 
period  during  which  the  foetal  hones  are  forming,  and  i;i 
diseases  of  the  kidneys,  because  of  non-elimination. 

Experiments. 

1.  Formation  of  "  Triple  Phosphate." — Place  some  urine 
in  a  beaker,  render  it  alkaline  with  ammonium  hydroxide,  add 
a  small  amount  of  magnesium  sulphate  solution  and  allow 
the  beaker  to  stand  in  a  cool  place  over  night.  Crystals  of 
ammonium  magnesium  phosphate,  "triple  phosphate,"  form 
under  these  conditions.  Examine  the  crystalline  sediment 
under  the  microscope  and  compare  the  forms  of  the  crystals 
with  those  shown  in  Fig.  96,  page  278. 

2.  "  Triple  Phosphate  "  Crystals  in  Ammoniacal  Fer- 
mentation.— Stand  some  urine  aside  in  a  beaker  for  several 
days.  Ammoniacal  fermentation  will  develop  and  "  triple 
phosphate"  crystals  will  form.     Examine  the  sediment  under 

1  In  metabolism  experiments,  a  statement  showing  the  relation  existing 
between  the  nitrogen  content  of  the  food  on  the  one  hand  and  that  of 
the  urine  and  feces  on  the  other,  for  a  definite  period,  is  termed  a  nitrogen 
balance  or  a  "  balance  of  the  income  and  outgo  of  nitrogen." 


278 


PHYSIOLOGICAL    CHEMISTRY. 
Fig.  96. 


"  Triple  Phosphate."     (Ogden.) 

the  microscope  and  compare  the  crystals  with  those  shown  in 
Fig.  96,  above. 

3.  Detection  of  Earthy  Phosphates. — Place  10  c.c.  of 
urine  in  a  test-tube  and  render  it  alkaline  with  ammonium 
hydroxide.  Warm  the  mixture  and  note  the  separation  of 
a  precipitate  of  earthy  phosphates. 

4.  Detection  of  Alkaline  Phosphates. — Filter  off  the 
earthy  phosphates  as  formed  in  the  last  experiment,  and  add 
a  small  amount  of  magnesia  mixture  (see  page  270)  to  the 
filtrate.  Now  warm  the  mixture  and  observe  the  formation  of 
a  white  precipitate  due  to  the  presence  of  alkaline  phosphates. 
Note  the  difference  in  the  size  of  the  precipitates  of  the  two 
forms  of  phosphates  from  this  same  volume  of  urine.  Which 
form  of  phosphates  were  present  in  the  larger  amount,  earthy 
or  alkaline? 

5.  Influence  upon  Fehling's  Solution. — Place  2  c.c.  of 
Fehling's  solution  in  a  test-tube,  dilute  it  with  4  volumes  of 
water  and  heat  to  boiling.  Add  a  solution  of  sodium  di- 
hydrogen  phosphate,  NaH2P04,  a  small  amount  at  a  time, 
and  heat  after  each  addition.  What  do  you  observe?  What 
does  this  observation  force  you  to  conclude  regarding  the 
interference  of  phosphates  in  the  testing  of  diabetic  urine  by 
means  of  Fehling's  test? 


URINE.  279 

Sodium  and  Potassium. 

The  elements  sodium  and  potassium  arc  always  present  in 
the  urine.  Usually  they  are  combined  with  such  acidic  radi- 
cals as  CI,  CO.j,  S04  and  PO.,.  The  amount  of  potassium,  ex- 
pressed as  l\j<  >.  excreted  in  -'4  hours  by  an  adult,  subsisting 

upon  a  mixed  diet,  is  on  the  average  2-3  grams,  whereas  the 
amount  of  sodium,  expressed  as  Na20,  under  the  same  condi- 
tions, is  ordinarily  4  6  grams.  The  ratio  of  K  to  Xa  is  gen- 
erally about"3:5.  The  absolute  quantity  of  these  elements 
excreted,  depends,  of  course,  in  large  measure,  upon  the  nature 
of  the  diet.  Because  of  the  non-ingestion  of  NaCl  and  the 
accompanying  destruction  of  potassium-containing  body  tis- 
sues, the  urine  during  fasting  contains  more  potassium  salts 
than  sodium  salts. 

Pathologically  the  output  of  potassium,  in  its  relation  to 
sodium,  may  be  increased  during  fever;  following  the  crisis, 
however,  the  output  of  this  element  may  be  decreased.  It 
may  also  be  increased  in  conditions  associated  with  acid 
intoxication. 

Calcium  and  Magnesium. 

The  greater  part  of  the  calcium  and  magnesium  excreted  in 
the  urine  is  in  the  form  of  phosphates.  The  daily  output,  which 
depends  principally  upon  the  nature  of  the  diet,  aggregates  on 
the  average  about  1  gram  and  is  made  up  of  the  phosphates  of 
calcium  and  magnesium  in  the  proportion  1  :  2.  The  percent- 
age of  calcium  salts  present  in  the  urine  at  any  one  time  forms 
no  dependable  index  as  to  the  absorption  of  this  class  of  salts, 
since  they  are  again  excreted  into  the  intestine  after  absorp- 
tion. It  is  therefore  impossible  to  draw  any  satisfactory  con- 
clusions regarding  the  excretion  of  the  alkaline  earths  unless 
we  obtain  accurate  analytical  data  from  both  the  feces  and 
the  urine. 

Very  little  is  known  positively  regarding  the  actual  course 
of  the  excretion  of  the  alkaline  earths  under  pathological 
conditions  except  that  an  excess  of  calcium  is  found  in  acid 
intoxication  and  some  diseases  of  the  bones. 


28o  PHYSIOLOGICAL    CHEMISTRY. 

Carbonates. 

Carbonates  generally  occur  in  small  amount  in  the  urine 
of  man  and  carnivora  under  normal  conditions,  whereas 
much  larger  quantities  are  ordinarily  present  in  the  urine  of 
herbivora.  The  alkaline  reaction  of  the  urine  of  herbivora 
is  dependable  in  great  measure  upon  the  presence  of  carbo- 
nates. In  general  a  urine  containing  carbonates  in  appre- 
ciable amount  is  turbid  when  passed  or  becomes  so  shortly 
after.  These  bodies  ordinarily  occur  as  alkali  or  alkaline 
earth  compounds  and  the  turbid  character  of  urine  contain- 
ing them  is  usually  due  principally  to  the  latter  class  of  sub- 
stances. The  carbonates  of  the  alkaline  earths  are  often 
found  in  amorphous  urinary  sediments. 

Iron. 

Iron  is  present  in  small  amount  in  normal  urine.  It  prob- 
ably occurs  partly  in  inorganic  and  partly  in  organic  combi- 
nation. The  iron  contained  in  urinary  pigments  or  chromo- 
gens  is  in  organic  combination.  According  to  different  in- 
vestigators the  iron  content  of  normal  urine  varies  from  0.012 
gram  to  0.15  gram  per  day. 

Experiment. 
Detection  of  Iron  in  Urine. — Evaporate  a  convenient  vol- 
ume (10-15  c.c.)  of  urine  to  dryness.  Incinerate  and  dissolve 
the  residue  in  a  few  drops  of  iron-free  hydrochloric  acid  and 
dilute  the  acid  solution  with  5  c.c.  of  water.  Divide  the  acid 
solution  into  two  parts  and  make  the  following  tests:  (a)  To 
the  first  part  add  a  solution  of  ammonium  sulphocyanide ;  a  red 
color  indicates  the  presence  of  iron,  (b)  To  the  second  part 
of  the  solution  add  a  little  potassium  ferrocyanide  solution;  a 
precipitate  of  Prussian  blue  forms  upon  standing. 

Fluorides,  Nitrates,  Silicates  and  Hydrogen  Peroxide. 

These  substances  are  all  found  in  traces  in  human  urine 
under   normal    conditions.     Nitrates   are   undoubtedly    intro- 


URINE.  28] 

duced  into  the  organism  in  the  water  ;m<l  ingested  food.  The 
average  excretion  of  nitrates  is  about  0.5  gram  per  day,  the 
output  being  the  large>t  upon  a  vegetable  diet  and  smallest 
upon  a  meat  diet.  Nitrites  are  found  only  in  urine  which  is 
undergoing  decomposition  and  are  formed  from  the  nitrates 
in  the  course  of  ammoniacal  fermentation.  Hydrogen  per- 
oxide has  been  detected  in  the  urine,  but  its  presence  is  be- 
lieved to  possess  no  pathological  importance. 


CHAPTER    XVIII. 
URINE:   PATHOLOGICAL  CONSTITUENTS.1 


Proteids 


Dextrose. 

Serum  albumin. 
Serum  globulin. 

f  Deutero-proteose. 
Proteoses  -j  Hetero-proteose. 

I "  Bence- Jones'  proteid." 
Peptone. 
Nucleo-proteid. 
Fibrin. 
Haemoglobin. 

Blood  {Form  elements. 
L  Pigment. 

Bile    (K^ents. 

Acetone. 

Diacetic  acid. 

/?-Oxybutyric  acid. 

Conjugate  glycuronates. 

Pentoses. 

Fat. 

Hsematoporphyrin. 

Lactose. 

Laevulose. 

Inosit. 

Laiose. 

Melanin. 

Urorosein. 

Unknown  substances. 

DEXTROSE. 

Traces  of  this  sugar  occur  in  normal  urine,  but  the  amount 
is  not  sufficient  to  be  readily  detected  by  the  ordinary  simple 

1  See  note  at  the  bottom  of  page  237. 

282 


URINE.  283 

qualitative  tests.  There  are  two  distinct  types  of  pathological 
glycosuria,  i.  <\,  transitory  glycosuria  and  persistent  glyco- 
suria. The  transitory  type  may  follow  the  ingestion  of  an 
excess  of  sugar,  causing  the  assimilation  limit  to  be  exceeded, 
or  it  may  accompany  any  one  of  several  disorders  which  cause 
an  impairment  of  the  power  of  assimilating  sugar.  In  the 
persistent  type  large  amounts  of  sugar  are  excreted  daily  in 
the  urine  for  long  periods  of  time.  Under  such  circumstances 
a  condition  known  as  diabetes  mellitus  exists.  Ordinarily, 
diabetic  urine  which  contains  a  high  percentage  of  sugar  pos- 
sesses a  faint  yellow  color,  a  high  specific  gravity  and  a  volume 
which  is  above  normal. 

Experiments. 

1.  Phenylhydrazin  Reaction. — Test  the  urine  according 
to  one  of  the  following  methods:  (a)  To  a  small  amount  of 
phenylhydrazin  mixture,  furnished  by  the  instructor,1  add 
5  c.c.  of  the  urine,  shake  well  and  heat  on  a  boiling  water- 
bath  for  one-half  to  three-quarters  of  an  hour.  Allow  the 
tube  to  cool  slowly  and  examine  the  crystals  microscopically 
(Plate  III.,  opposite  page  5).  If  the  solution  has  become  too 
concentrated  in  the  boiling  process  it  will  be  light-red  in  color 
and  no  crystals  will  separate  until  it  is  diluted  with  water. 

Yellow  crystalline  bodies  called  osazons  are  formed  from 
certain  sugars  under  these  conditions,  each  individual  sugar 
giving  rise  to  an  osazon  of  a  definite  crystalline  form  which 
is  typical  for  that  sugar.  Each  osazon  has  a  definite  melting- 
point,  and  as  a  further  and  more  accurate  means  of  identi- 
fication it  may  be  recrystallized  and  identified  by  the  determi- 
nation of  its  melting-point  and  nitrogen  content.  The  reac- 
tion taking  place  in  the  formation  of  phenyldextr  osazon  is 
as  follows : 

1  This  mixture  is  prepared  by  combining  one  part  of  phenylhydrazin- 
hydrochloride  and  two  parts  of  sodium  acetate,  by  weight.  These  are 
thoroughly  mixed  in  a  mortar. 


284  PHYSIOLOGICAL    CHEMISTRY. 

C6H1206  +  2(H2N*NH-  C6H5)  = 

Dextrose.  Phenylhydrazin. 

C6H1004(N-  NH-  C6H5)2  +  2H20  +  H2. 

Phenyl  dextrosazon. 

(b)  Place  5  c.c.  of  the  urine  in  a  test-tube,  add  1  c.c.  of 
phenylhydrazin-acetate  solution  furnished  by  the  instruc- 
tor,1 and  heat  on  a  boiling  water-bath  for  one-half  to  three- 
quarters  of  an  hour.  Allow  the  liquid  to  cool  slowly  and 
examine  the  crystals  microscopically  (Plate  III.,  opposite  p.  5). 

The  phenylhydrazin  test  has  been  so  modified  by  Cipollina 
as  to  be  of  use  as  a  rapid  clinical  test.  The  directions  for 
this  test  are  given  in  the  next  experiment. 

2.  Cipollina's  Test. — Thoroughly  mix  4  c.c.  of  urine,  5 
drops  of  phenylhydrazin  (the  base)  and  one-half  c.c.  of  glacial 
acetic  acid  in  a  test-tube.  Heat  the  mixture  for  about  one 
minute  over  a  low  flame,  shaking  the  tube  continually  to  pre- 
vent loss  of  fluid  by  bumping.  Add  4-5  drops  of  potassium 
hydroxide  or  sodium  hydroxide  (sp.  gr.  1.16),  being  certain 
that  the  fluid  in  the  test-tube  remains  acid;  heat  the  mixture 
again  for  a  moment  and  then  cool  the  contents  of  the  tube. 
Ordinarily  the  crystals  form  at  once,  especially  if  the  urine 
possesses  a  low  specific  gravity.  If  they  do  not  appear  imme- 
diately allow  the  tube  to  stand  at  least  20  minutes  before 
deciding  upon  the  absence  of  sugar. 

Examine  the  crystals  under  the  microscope  and  compare 
them  with  those  shown  in  Plate  III.,  opposite  page  5. 

3.  Reduction  Tests. — To  their  aldehyde  or  ketone  struc- 
ture many  sugars  owe  the  property  of  readily  reducing  the 
alkaline  solutions  of  the  oxides  of  metals  like  copper,  bismuth 
and  mercury;  they  also  possess  the  property  of  reducing 
ammoniacal  silver  solutions  with  the  separation  of  metallic 
silver.  Upon  this  property  of  reduction  the  most  widel)r  used 
tests  for  sugars  are  based.    When  whitish-blue  cupric  hydrox- 

1  This  solution  is  prepared  by  mixing  one  part  by  volume,  in  each  case, 
of  glacial  acetic  acid,  one  part  of  water  and  two  parts  of  phenylhydrazin 
(the  base). 


URINE. 


J.S. 


ide  in  suspension  in  an  alkaline  liquid  is  heated  it  is  convi 
into  insoluble  black  cupric  oxide,  but  if  a  reducing  agent  like 
certain  sugars  be  present  the  cupric  hydroxide  is  reduced  to 
insoluble  yellow  cuprous  hydroxide,  which  in  turn  on  further 
heating  may  be  converted  into  brownish-red  or  red  cuprous 
oxide.    These  changes  are  indicated  as  follows: 


Cu 


/ 

i 

\ 


OH 


OH 

Cupric  hydroxide, 
(whitish-blue). 


Cu 


Cu 


/ 
i 

\ 

/ 
i 

\ 


OH 

OH 
OH 

OH 


0u=0  +  IT,0. 


Cupric  oxide, 
(black). 


2Cu-OH  +  H20-f  0. 


Cuprous  hydroxide, 
(yellow). 


Cu-OH 
Cu-OH 

Cuprous  hydroxide 
(yellow). 


Cu 


\ 

( 

/ 


0  +  H20. 


Cu 

Cuprous  oxide, 
(brownish-red). 


The  chemical  equations  here  discussed  are  exemplified  in 
Trommer's  and  Fehling's  tests. 

(a)  Trommer's  Test. — To  5  c.c.  of  urine  in  a  test-tube 
add  one-half  its  volume  of  KOH  or  NaOH.  Mix  thoroughly 
and  add,  drop  by  drop,  agitating  after  the  addition  of  each 
drop,  a  very  dilute  solution  of  cupric  sulphate.  Continue 
the  addition  until  there  is  a  slight  permanent  precipitate  of 


286  PHYSIOLOGICAL    CHEMISTRY. 

cupric  hydroxide  and  in  consequence  the  solution  is  slightly 
turbid.  Heat,  and  the  cupric  hydroxide  is  reduced  to  yellow 
cuprous  hydroxide  or  to  brownish-red  cuprous  oxide.  If  the 
solution  of  cupric  sulphate  used  is  too  strong,  a  small  brown- 
ish-red precipitate  produced  in  the  presence  of  a  low  percent- 
age of  dextrose  may  be  entirely  masked.  On  the  other  hand,  if 
too  little  cupric  sulphate  is  used  a  light-colored  precipitate 
formed  by  uric  acid  and  purin  bases  may  obscure  the  brownish- 
red  precipitate  of  cuprous  oxide.  The  action  of  KOH  or 
NaOH  in  the  presence  of  an  excess  of  sugar  and  insufficient 
copper  will  produce  a  brownish  color.  Phosphates  of  the 
alkaline  earths  may  also  be  precipitated  in  the  alkaline  solution 
and  be  mistaken  for  cuprous  hydroxide.  Trommer's  test  is 
not  very  satisfactory. 

(b)  Fehling's  Test. — To  about  i  c.c.  of  Fehling's  solu- 
tion1 in  a  test-tube  add  about  4  c.c.  of  water,  and  boil.  This 
is  done  to  determine  whether  the  solution  will  of  itself  cause 
the  formation  of  a  precipitate  of  brownish-red  cuprous  oxide. 
If  such  a  precipitate  forms,  the  Fehling's  solution  must  not 
be  used.  Add  urine  to  the  warm  Fehling's  solution,  a  few 
drops  at  a  time,  and  heat  the  mixture  after  each  addition. 
The  production  of  yellow  cuprous  hydroxide  or  brownish- 
red  cuprous  oxide  indicates  that  reduction  has  taken  place. 
The  yellow  precipitate  is  more  likely  to  occur  if  the  urine  is 
added  rapidly  and  in  large  amount,  whereas  with  a  less  rapid 
addition  of  smaller  amounts  of  urine  the  brownish-red  pre- 
cipitate is  generally  formed. 

This  is  a  much  more  satisfactory  test  than  Trommer's,  but 

1  Fehling's  solution  is  composed  of  two  definite  solutions — a  cupric 
sulphate  solution  and  an  alkaline  tartrate  solution,  which  may  be  prepared 
as  follows : 

Cupric  sulphate  solution  =  34.64  grams  of  cupric  sulphate  dissolved  in 
water  and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =  125  grams  of  potassium  hydroxide  and  173 
grams  of  Rochelle  salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bot- 
tles and  mixed  in  equal  volumes  when  needed  for  use.  This  is  done  to 
prevent  deterioration. 


URINE.  287 

even  this  test  is  not  entirely  reliable  when  used  to  detect  sugar 
in  the  urine.  Such  bodies  as  conjugate  glycuronates,  uric  acid, 
nucleo-proteid  and  homogentisk  acid,  when  present  in  suffi- 
cient ann 'tint,  may  produce  a  result  similar  to  that  produced  by 
sugar.  Phosphates  of  the  alkaline  earths  may  be  precipitated 
by  the  alkali  of  the  Fehling's  solution  and  in  appearance  may  be 
mistaken  for  the  cuprous  hydroxide.  Cupric  hydroxide  may 
also  be  reduced  to  cuprous  oxide  and  this  in  turn  be  dissolved 
by  creatinin,  a  normal  urinary  constituent.  This  will  give 
the  urine  under  examination  a  greenish  tinge  and  may  obscure 
the  sugar  reaction  even  when  a  considerable  amount  of  sugar 
is  present. 

(c)  Allen's  Modification  of  Fehling's  Test. — The  fol- 
lowing procedure  is  recommended:  "From  7  to  8  c.c.  of  the 
sample  of  urine  to  be  tested  is  heated  to  boiling  in  a  test-tube, 
and,  without  separating  any  precipitate  of  albumin  which  may 
be  produced,  5  c.c.  of  the  solution  of  cupric  sulphate  used  for 
preparing  Fehling's  solution  is  added.  This  produces  a  pre- 
cipitate containing  uric  acid,  xanthin,  hypoxanthin,  phos- 
phates, etc.  To  render  the  precipitation  complete,  however, 
it  is  desirable  to  add  to  the  liquid,  when  partially  cooled,  from 
1  to  2  c.c.  of  a  saturated  solution  of  sodium  acetate  having 
a  feebly  acid  reaction  to  litmus.1  The  liquid  is  filtered  and 
to  the  filtrate,  which  will  have  a  bluish-green  color,  5  c.c. 
of  the  alkaline  tartrate  mixture  used  for  preparing  Fehling's 
solution  is  added,  and  the  liquid  boiled  for  15-20  sec- 
onds. In  the  presence  of  more  than  0.25  per  cent  of  sugar, 
separation  of  cuprous  oxide  occurs  before  the  boiling-point 
is  reached;  but  with  smaller  quantities  precipitation  takes 
place  during  the  cooling  of  the  solution,  which  becomes  green- 

1  Sufficient  acetic  acid  should  be  added  to  the  sodium  acetate  solution 
to  render  it  feebly  acid  to  litmus.  A  saturated  solution  of  sodium  acetate 
keeps  well,  but  weaker  solutions  are  apt  to  become  mouldy,  and  then 
possess  the  power  of  reducing  Fehling's  solution.  Hence  it  is  essential 
in  all  cases  of  importance  to  make  a  blank  test  by  mixing  equal  measures 
of  cupric  sulphate  solution,  alkaline  tartrate  solution  and  water,  adding  a 
little  sodium  acetate  solution,  and  heating  the  mixture  to  boiling. 


2b»  PHYSIOLOGICAL    CHEMISTRY. 

ish.  opaque,  and  suddenly  deposits  cuprous  oxide  as  a  fine 
br<  »wnish-red  precipitate." 

(d)  Boettger's  Test. — To  5  c.c.  of  urine  in  a  test-tube 
add  1  c.c.  of  KOH  or  NaOH  and  a  very  small  amount  of  bis- 
muth subnitrate,  and  boil.  The  solution  will  gradually  darken 
and  finally  assume  a  black  color  due  to  reduced  bismuth.  If  the 
test  is  made  with  urine  containing  albumin  this  must  be  re- 
moved, by  boiling  and  filtering,  before  applying  the  test,  since 
with  albumin  a  similar  change  of  color  is  produced  (bismuth 
sulphide). 

(e)  Nylander's  Test  (Almen's  Test). — To  5  c.c.  of  urine 
in  a  test-tube  add  one-tenth  its  volume  of  Nylander's  reagent1 
and  boil  two  or  three  minutes.  The  mixture  will  darken  if 
reducing  sugar  is  present  and  upon  standing  for  a  few  mo- 
ments a  black  color  will  appear.  This  color  is  due  to  the 
precipitation  of  bismuth.  If  the  test  is  made  on  urine  con- 
taining albumin  this  must  be  removed,  by  boiling  and  filtering, 
before  applying  the  test.  It  is  claimed  by  Bechold  that  Ny- 
lander's and  Boettger's  tests  give  a  negative  reaction  with 
solutions  containing  sugar  when  mercuric  chloride  or  chloro- 
form is  present,  a  claim  which  has  very  recently  been  contra- 
dicted by  Zeidlitz. 

A  positive  Nylander  or  Boettger  test  is  probably  due  to 
the  following  reactions : 

(a)  Bi(OH)2N03  +  KOH  =  Bi(0H)8  +  KN03. 

(b)  2Bi(OH)3  —  30  =  Bi2  +  3H20. 

4.  Fermentation  Test. — Rub  up  in  a  mortar  about  15  c.c. 
of  the  urine  with  a  small  piece  of  compressed  yeast.  Trans- 
fer the  mixture  to  a  saccharometer  (Fig.  2,  p.  10)  and  stand 
it  aside  in  a  warm  place  for  about  12  hours.  If  dextrose  is 
present,  alcoholic  fermentation  will  occur  and  carbon  dioxide 
will  collect  as  a  gas  in  the  upper  portion  of  the  tube.     On  the 

1  Nylander's  reagent  is  prepared  by  digesting  2  grams  of  bismuth  sub- 
nitrate  and  4  grams  of  Rochelle  salt  in  100  c.c.  of  a  10  per  cent  potassium 
hydroxide  solution.     The  reagent  is  then  cooled  and  filtered. 


URINE.  289 

completion  of  fermentation  introduce,  by  means  of  a  bent 
pipette,  a  little  K(  )l  I  solution  into  the  graduated  portion,  place 
the  thumb  tightly  over  the  opening  in  the  apparatus  and 
invert  the  saccharometer.     Explain  the  result. 

5.  Barfoed's  Test. — To  2-3  c.c.  of  Barfoed's  solution1  in 
a  test-tube  add  a  few  drops  of  urine  and  boil.  Allow  the  tube 
to  stand  a  few  minutes  ami  examine.  In  the  presence  of  dex- 
tiose  a  red  precipitate  forms.      What  is  it? 

6.  Polariscopic  Examination. — For  directions  as  to  the 
use  of  the  polariscope  see  page  1  1. 

PROTEIDS. 

Normal  urine  contains  a  trace  of  proteid  material  but  the 
amount  present  is  so  slight  as  to  escape  detection  by  any  of 
the  simple  tests  in  general  use  for  the  detection  of  proteid 
Urinary  constituents.  The  following  are  the  more  important 
forms  of  proteid  material  which  have  been  detected  in  the 
urine  under  pathological  conditions  : 

( 1 )  Serum  albumin. 

(2)  Serum  globulin. 

f  Deutero-proteose. 

(3)  Proteoses  ">  Hetero-proteose. 

I "  Bence- Jones'  proteid." 

(4)  Peptone. 

(5)  Nucleo-proteid. 

(6)  Fibrin. 

(7)  Haemoglobin. 

ALBUMIN. 

Albuminuria  is  a  condition  in  which  serum  albumin  or 
serum  globulin  appears  in  the  urine.  There  are  two  distinct 
forms  of  albuminuria,  i.  c,  renal  albuminuria  and  accidental 
albuminuria.     Sometimes  the  terms  "  true  "  albuminuria  and 

barfoed's  solution  is  prepared  as  follows:  Dissolve  4  grams  of  cupric 
acetate  in  100  c.c.  of  water  and  acidify  with  acetic  acid. 
20 


29O  PHYSIOLOGICAL    CHEMISTRY. 

"  false  "  albuminuria  are  substituted  for  those  just  given.  In 
the  renal  type  the  albumin  is  excreted  by  the  kidneys.  This 
is  the  more  serious  form  of  the  malady  and  at  the  same  time 
is  more  frequently  encountered  than  the  accidental  type. 
Among  the  causes  of  renal  albuminuria  are  altered  blood  pres- 
sure in  the  kidneys,  altered  kidney  structure,  or  changes  in 
the  composition  of  the  blood  entering  the  kidneys,  thus  allow- 
ing the  albumin  to  diffuse  more  readily.  In  the  accidental 
form  of  albuminuria  the  albumin  is  not  excreted  by  the  kid- 
neys as  is  the  case  in  the  renal  form  of  the  disorder,  but  arises 
from  the  blood,  lymph  or  some  albumin-containing  exudate 
coming  into  contact  with  the  urine  at  some  point  below  the 

kidneys. 

Experiments. 

1.  Heller's  Ring  Test. — Place  5  c.c.  of  concentrated  HN03 
in  a  test-tube,  incline  the  tube,  and,  by  means  of  a  pipette 
allow  the  urine  to  flow  slowly  down  the  side.1  The  liquids 
should  stratify  with  the  formation  of  a  white  zone  of  precip- 
itated albumin  at  the  point  of  juncture.  If  the  albumin  is 
present  in  very  small  amount  the  white  zone  may  not  form 
until  the  tube  has  been  allowed  to  stand  for  several  minutes. 
If  the  urine  is  quite  concentrated  a  white  zone,  due  to  uric 
acid  or  urates,  will  form  upon  treatment  with  nitric  acid  as 
indicated.  This  ring  may  be  easily  differentiated  from  the 
albumin  ring  by  repeating  the  test  after  diluting  the  urine 
with  3  or  4  volumes  of  water,  whereupon,  the  ring,  if  due 
to  uric  acid  or  urates,  will  not  appear.  It  is  ordinarily  pos- 
sible to  differentiate  between  the  albumin  ring  and  the  uric 
acid  ring  without  diluting  the  urine,  since  the  ring,  when 
due  to  uric  acid,  has  ordinarily  a  less  sharply  defined  upper 
border,  is  generally  broader  than  the  albumin  ring  and  fre- 
quently is  situated  in  the  urine  above  the  point  of  contact 
with  the  nitric  acid.  Concentrated  urines  also  occasionally  ex- 
hibit the  formation,  at  the  point  of  contact,  of  a  crystalline 

1  An  apparatus  called  the  albumoscope  has  been  devised  for  use  in  this 
test  and  has  met  with  considerable  favor. 


URINE.  291 

ring  with  very  sharply  defined  borders.  This  is  urea  nitrate 
and  is  easily  distinguished  from  the  "  fluffy  "  ring  of  albumin. 
If  there  is  any  difficulty  in  differentiation  a  simple  dilution  of 
the  urine  with  water,  as  above  described,  will  remove  the  diffi- 
culty. Various  colored  zones,  due  either  to  the  presence  of 
indican,  bile  pigments  or  to  the  oxidation  of  other  organic  uri- 
nary constituents,  may  form  in  this  test  under  certain  condi- 
tion^. These  colored  rings  should  never  be  confounded  with 
the  white  ring  which  alone  denotes  the  presence  of  albumin. 
\  fter  the  administration  of  certain  drugs  a  white  precipi- 
tate of  resin  acids  may  form  at  the  point  of  contact  of  the 
two  fluids  and  may  cause  the  observer  to  draw  wrong  conclu- 
sions. This  ring,  if  composed  of  resin  acids,  will  dissolve 
in  alcohol,  whereas  the  albumin  ring  will  not  dissolve. 

2.  Roberts'  Ring  Test. — Place  5  c.c.  of  Roberts'  reagent1 
in  a  test-tube,  incline  the  tube,  and,  by  means  of  a  pipette,  al- 
low the  urine  to  flow  slowly  down  the  side.  The  liquids  should 
stratify  with  the  formation  of  a  white  zone  of  precipitated 
albumin  at  the  point  of  juncture.  This  test  is  a  modification 
of  Heller's  ring  test  and  is  rather  more  satisfactory  than  that 
test,  since  the  colored  rings  never  form  and  the  consequent 
confusion  is  avoided. 

3.  Spiegler's  Ring  Test. — Place  5  c.c.  of  Spiegler's  rea- 
gent2 in  a  test-tube,  incline  the  tube,  and,  by  means  of  a  pipette, 
allow  5  c.c.  of  urine,  acidified  with  acetic  acid,  to  flow  slowly 
down  the  side.  A  white  zone  will  form  at  the  point  of 
contact.  This  is  an  exceedingly  delicate  test,  in  fact,  too  deli- 
cate for  ordinary  clinical  purposes,  since  it  serves  to  detect 
albumin  when  present  in  the  merest  trace  ( 1  :  250,000)  and 
hence  most  normal  urines  will  give  a  positive  reaction  for 
albumin  when  this  test  is  applied. 

Roberts'  reagent  is  composed  of  1  volume  of  concentrated  HNO3  and  5 
volumes  of  a  saturated  solution  of  MgSGv 

2 Spiegler's  reagent  has  the  following  composition: 

Tartaric  acid 20  grams. 

Mercuric  chloride 40  grams. 

Glycerin    100  grams. 

Distilled  water 1000  grams. 


292  PHYSIOLOGICAL    CHEMISTRY. 

Some  investigators  claim  that  the  delicacy  of  this  test  de- 
pends upon  the  presence  of  sodium  chloride  in  the  urine,  the 
test  losing  accuracy  if  the  sodium  chloride  content  be  low. 

4.  Jolles'  Reaction. — Shake  5  c.c.  of  urine  with  1  c.c.  of 
30  per  cent  acetic  acid  and  4  c.c.  of  Jolles'  reagent1  in  a  test- 
tube.     A  white  precipitate  indicates  the  presence  of  albumin. 

Care  should  be  taken  to  use  the  correct  amount  of  acetic  acid, 
since  the  use  of  too  small  an  amount  may  result  in  the  forma- 
tion of  mercury  combinations  which  may  cause  confusion. 
In  the  presence  of  iodine,  mercuric  iodide  will  form  but  may 
readily  be  differentiated  from  albumin  through  the  fact  that 
it  is  soluble  in  alcohol. 

5.  Coagulation  or  Boiling  Test. —  (a)  Heat  5  c.c.  of  urine 
to  boiling  in  a  test-tube.  A  precipitate  forming  at  this  point 
is  due  either  to  albumin  or  to  phosphates.  Acidify  the  urine 
slightly  by  the  addition  of  3-5  drops  of  very  dilute  acetic  acid, 
adding  the  acid  drop  by  drop  to  the  hot  solution.  If  the  pre- 
cipitate is  due  to  phosphates  it  will  disappear  under  these  con- 
ditions, whereas  if  it  is  due  to  albumin  it  will  not  only  fail  to 
disappear  but  will  become  more  flocculent  in  character,  since 
the  reaction  of  a  fluid  must  be  acid  to  secure  the  complete  pre- 
cipitation of  the  albumin  by  this  coagulation  process.  Too 
much  acid  should  be  avoided  since  it  will  cause  the  albumin  to 
go  into  solution.  Certain  resin  acids  may  be  precipitated  by 
the  acid,  but  the  precipitate  due  to  this  cause  may  be  easily 
differentiated  from  the  albumin  precipitate  by  reason  of  its 
solubility  in  alcohol. 

(b)  A  modification  of  this  test  in  quite  general  use  is  as 
follows :  Fill  a  test-tube  two-thirds  full  of  urine  and  gently 
heat  the  upper  half  of  the  fluid  to  boiling,  being  careful  that 
this  fluid  does  not  mix  with  the  lower  half.  A  turbidity  indi- 
cates albumin  or  phosphates.     Acidify  the  urine  slightly  by 

1  Jolles'  reagent  has  the  following  composition : 

Succinic  acid 40  grams. 

Mercuric   chloride '. 20  grams. 

Sodium  chloride 20  grams. 

Distilled   water 1000  grams. 


URINE.  293 

the  addition  of  3  5  drops  of  dilute  acetic  acid,  when  the  tur- 
bidity, it"  due  to  phosphates,  will  disappear. 

Nitric  acid  is  often  used  in  place  of  acetic  acid  in  these  t< 
In  case  nitric  arid  is  used  ordinarily   1    2  drops  is  sufficient. 

6.  Acetic  Acid  and  Potassium  Ferrocyanide  Test. — To 
5  C.C.  of  urine  in  a  test-tube  add  5  [O  drops  of  acetic  acid. 
Mix  well  and  add  potassium  ferrocyanide  drop  by  drop,  until  a 
precipitate  f<  >rms. 

7.  Tanret's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add 
Tanret's  reagent1  drop  by  drop  until  a  turbidity  or  precipitate 
forms.  This  is  an  exceedingly  delicate  test.  Sometimes  the 
urine  is  stratified  upon  the  reagent  as  in  Heller's  or  Roberts' 
ring"  tests. 

8.  Sodium  Chloride  and  Acetic  Acid  Test. — Mix  two 
volumes  of  urine  and  one  volume  of  a  saturated  solution  of 
sodium  chloride  in  a  test-tube,  acidify  with  acetic  acid  and  heat 
to  boiling.  The  production  of  a  cloudiness  or  the  formation 
of  a  precipitate  indicates  the  presence  of  albumin.  The  resin 
acids  may  interfere  here  as  in  the  ordinary  coagulation  test 
1  page  292 )  but  they  may  be  easily  differentiated  from  albumin 
by  means  of  their  solubility  in  alcohol. 

GLOBULIN. 

Serum  globulin  is  not  a  constituent  of  normal  urine  but 
frequently  occurs  in  the  urine  under  pathological  conditions  and 
is  ordinarily  associated  with  serum  albumin.  In  albuminuria 
globulin  in  varying  amounts  often  accompanies  the  albumin, 
and  the  clinical  significance  of  the  two  is  very  similar.  Under 
certain  conditions  globulin  may  occur  in  the  urine  unaccom- 
panied by  albumin. 

Experiments. 

Globulin  will   respond  to  all  the  tests  just  outlined  under 

Albumin.     If  it  is  desirable  to  differentiate  between  albumin 

'Tanret's  reagent  is  prepared  as  follows:  Dissolve  1.35  gram  of  mercuric 
chloride  in  25  c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium 
iodide  dissolved  in  25  c.c.  of  water,  then  make  the  total  solution  up  to  60  c.c. 
with  water  and  add  20  c.c.  of  glacial  acid  to  the  mixture. 


294  PHYSIOLOGICAL    CHEMISTRY. 

and  globulin  in  any  urine  the  following  processes  may  be 
employed : 

i.  Saturation  With  Magnesium  Sulphate. — Place  25  c.c. 
of  neutral  urine  in  a  small  beaker  and  add  pulverized  mag- 
nesium sulphate  in  substance  to  the  point  of  saturation.  If  the 
proteid  present  is  globulin  it  will  precipitate  at  this  point.  If 
no  precipitate  is  produced  acidify  the  saturated  solution  with 
acetic  acid  and  warm  gently.  Albumin  will  be  precipitated 
if  present. 

The  above  procedure  may  be  used  to  separate  globulin  and 
albumin  if  present  in  the  same  urine.  To  do  this  filter  off  the 
globulin  after  it  has  been  precipitated  by  the  magnesium  sul- 
phate, then  acidify  the  clear  solution  and  warm  gently  as 
directed.     Note  the  formation  of  the  albumin  precipitate. 

2.  Half-Saturation  With  Ammonium  Sulphate. — Place 
25  c.c.  of  neutral  urine  in  a  small  beaker  and  add  an  equal 
volume  of  a  saturated  solution  of  ammonium  sulphate.  Globu- 
lin, if  present,  will  be  precipitated.  If  no  precipitate  forms  add 
ammonium  sulphate  in  substance  to  the  point  of  saturation. 
If  albumin  is  present  it  will  be  precipitated  upon  saturation  of 
the  solution  as  just  indicated.  This  method  may  also  be  used 
to  separate  globulin  and  albumin  when  they  occur  in  the  same 
urine. 

Frequently  in  urine  which  contains  a  large  amount  of  urates 
a  precipitate  of  ammonium  urate  may  occur  when  the  am- 
monium sulphate  solution  is  added  to  the  urine.  This  urate 
precipitate  should  not  be  confounded  with  the  precipitate  due 
to  globulin.  The  two  precipitates  may  be  differentiated  by 
means  of  the  fact  that  the  urate  precipitate  ordinarily  appears 
only  after  the  lapse  of  several  minutes  whereas  the  globulin 
generally  precipitates  at  once. 

PROTEOSE  AND   PEPTONE. 

Proteoses,  particularly  deutero-proteose  and  hetero-proteose, 
have  frequently  been  found  in  the  urine  under  various  patho- 
logical  conditions   such   as   diphtheria,   pneumonia,   intestinal 


URINE.  29S 

ulcer,  carcinoma,  dermatitis,  osteomalacia,  atrophy  of  the  kid- 
neys and  in  sarcomata  of  the  bones  of  the  trunk.  "  Bence- 
Jones*  proteid,"  a  proteose-like  substance,  is  of  interest  in  this 
connection  and  it-  appearance  in  the  urine  is  believed  to  be 
of  great  diagnostic  importance  in  cases  of  multiple  myeloma 
or  myelogenic  osteosarcoma.  By  some  investigators  this  pro- 
teid is  held  to  be  a  variety  of  lietero-proteose  whereas  others 
claim  that  it  possesses  albumin  characteristics. 

Peptone  certainly  occurs  much  less  frequently  as  a  constitu- 
ent of  the  urine  than  does  proteose,  in  fact  most  investigators 
seriously  question  its  presence  under  any  conditions.  There 
are  many  instances  of  peptonuria  cited  in  the  early  literature 
but  because  of  the  uncertainty  in  the  conception  of  what  really 
constituted  a  peptone  it  is  probable  that  in  many  cases  of  so- 
called  peptonuria  the  proteid  present  was  really  proteose. 

Experiments. 

1.  Boiling  Test. — Make  the  ordinary  coagulation  test  ac- 
cording to  the  directions  given  under  Albumin,  page  292.  If 
no  coagulable  proteid  is  found  allow  the  boiled  urine  to  stand 
and  note  the  gradual  appearance,  in  the  cooled  fluid,  of  a  flaky 
precipitate  of  proteose.  This  is  a  crude  test  and  should  never 
be  relied  upon. 

2.  Schulte's  Method. — Acidify  50  c.c.  of  urine  with  dilute 
acetic  acid  and  filter  off  any  precipitate  of  nucleo-proteid  which 
may  form.  Now  test  a  few  cubic  centimeters  of  the  urine  for 
coagulable  proteid,  by  tests  2  and  5  under  Albumin,  pp.  291- 
292.  If  coagulable  proteid  is  present  remove  it  by  coagulation 
and  filtration  before  proceeding.  Introduce  25  c.c.  of  the  urine, 
freed  from  coagulable  proteid,  into  150  c.c.  of  absolute  alcohol 
and  allow  it  to  stand  for  12-24  hours.  Decant  the  supernatant 
fluid  and  dissolve  the  precipitate  in  a  small  amount  of  hot 
water.  Xow  filter  this  solution,  and  after  testing  again  for 
nucleo-proteid  with  very  dilute  acetic  acid,  try  the  biuret  test. 
If  this  test  is  positive  the  presence  of  proteose  is  indicated.1 

1  If  it  is  considered  desirable  to  test  for  peptone  the  proteose  may  be 
removed  by  saturation  with  (NH^SCX  according  to  the  directions  given 
on  page  59  and  the  tilt  rate  tested  for  peptone  by  the  biuret  test. 


296  PHYSIOLOGICAL    CHEMISTRY. 

Urobilin  does  not  ordinarily  interfere  with  this  test  since 
it  is  almost  entirely  dissolved  by  the  absolute  alcohol  when  the 
proteose  is  precipitated. 

3.  v.  Aldor's  Method. — Acidify  10  c.c.  of  urine  with 
hydrochloric  acid,  add  phosphotungstic  acid  until  no  more  pre- 
cipitate forms  and  centifugate1  the  solution.  Decant  the  super- 
natant fluid,  add  some  absolute  alcohol  to  the  precipitate  and 
centrifugate  again.  This  washing  with  alcohol  is  intended  to 
remove  the  urobilin  and  hence  should  be  continued  so  long  as 
the  alcohol  exhibits  any  coloration  whatever.  Now  suspend 
the  precipitate  in  water  and  add  potassium  hydroxide  to  bring 
it  into  solution.  At  this  point  the  solution  may  be  blue  in 
color  in  which  case  decolorization  may  be  secured  by  gently 
heating.  Apply  the  biuret  test  to  the  cool  solution.  A  posi- 
tive biuret  test  indicates  the  presence  of  proteoses. 

4.  Detection  of  "  Bence-Jones'  Proteid." — Heat  the  sus- 
pected urine  very  gently,  carefully  noting  the  temperature. 
At  as  low  a  temperature  as  400  C.  a  turbidity  may  be  observed 
and  as  the  temperature  is  raised  to  about  6o°  C.  a  flocculent 
precipitate  forms  and  clings  to  the  sides  of  the  test-tube.  If 
the  urine  is  now  acidified  very  slightly  with  acetic  acid  and  the 
temperature  further  raised  to  ioo°  C.  the  precipitate  at  least 
partly  disappears;  it  will  return  upon  cooling  the  tube. 

This  property  of  precipitating  at  so  low  a  temperature  and 
of  dissolving  at  a  higher  temperature  is  typical  of  "  Bence- 
Jones'  proteid  "  and  may  be  used  to  differentiate  it  from  all 
other  forms  of  proteid  material  occurring  in  the  urine. 

NUCLEO-PROTEID. 

There  has  been  considerable  controversy  as  to  the  proper 
classification  for  the  proteid  body  which  forms  the  "  nubecula  " 
of  normal  urine.  By  different  investigators  it  has  been  called 
mucin,  mucoid,  phospho-proteid,  nuclco-albumin  and  nucleo- 
proteid.     Of  course,  according  to  the  modern  acceptation  of 

1  If  not  convenient  to  use  a  centrifuge  the  precipitate  may  be  filtered  off 
and  washed  on  the  filter  paper  with  alcohol. 


URINE.  297 

the  meanings  of  these  terms  they  cannot  be  synonymous. 
Mucin  and  mucoid  are  glucoproteids  and  hence  contain  no 
phosphorus    (see  p.  61).   whereas  phospho-proteids,   nucleo- 

alhnmins  and  nncleo-proteids  are  phosphorized  bodies.  It  may 
possibly  be  that  both  these  forms  of  proteid.  i.  <\,  the  glucopro- 
teid  and  the  phosphorized  type,  occur  in  the  urine  under  certain 
conditions  (seepage  264).  In  this  connection  we  will  use  the 
term  nucleo-proteid.  The  pathological  conditions  under  which 
the  content  of  nucleo-proteid  is  increased  includes  all  affections 
of  the  urinary  passages  and  in  particular  pyelitis,  nephritis  and 
inflammation  of  the  bladder.  » 

Experiments. 

1.  Detection  of  Nucleo-proteid. — Place  10  c.c.  of  urine  in 
a  small  beaker,  dilute  it  with  three  volumes  of  water,  to  prevent 
precipitation  of  urates,  and  make  the  reaction  very  strongly 
acid  with  acetic  acid.  If  the  urine  becomes  turbid  it  is  an  indi- 
cation that  nucleo-proteid  is  present. 

If  the  urine  under  examination  contains  albumin  the  greater 
portion  of  this  substance  should  be  removed  by  boiling  the 
urine  before  testing  it  for  the  presence  of  nucleo-proteid. 

2.  Ott's  Precipitation  Test. — Mix  2^  c.c.  of  the  urine  with 
an  equal  volume  of  a  saturated  solution  of  sodium  chloride  and 
slowly  add  Almen's  reagent.1  In  the  presence  of  nucleo- 
proteid  a  voluminous  precipitate  forms. 

BLOOD. 

The  pathological  conditions  in  which  blood  occurs  in  the 
urine  may  be  classified  under  the  two  divisions  hematuria  and 
hemoglobinuria.  In  hematuria  we  are  able  to  detect  not  only 
the  haemoglobin  but  the  unruptured  corpuscles  as  well,  whereas 
in  hemoglobinuria  the  pigment  alone  is  present.  Hematuria 
is  brought  about  through  blood  passing  into  the  urine  because 
of  some  lesion  of  the  kidney  or  of  the  urinary  tract  below  the 

1  Dissolve  5  grams  of  tannin  in  240  c.c.  of  50  per  cent  alcohol  and  add 
10  c.c.  of  25  per  cent  acetic  acid. 


298  PHYSIOLOGICAL    CHEMISTRY. 

kidney.  Hemoglobinuria  is  brought  about  through  haemo- 
lysis, i.  c,  the  rupturing  of  the  stroma  of  the  erythrocyte  and 
the  liberation  of  the  haemoglobin.  This  may  occur  in  scurvy, 
typhus,  pyemia,  purpura  and  in  other  diseases.  It  may  also 
occur  as  the  result  of  a  burn  covering  a  considerable  area  of 
the  body,  or  may  be  brought  about  through  the  action  of  cer- 
tain poisons  or  by  the  injection  of  various  substances  having 
the  power  of  dissolving  the  erythrocytes.  Transfusion  of 
blood  may  also  cause  hemoglobinuria. 

Experiments. 

1.  Heller's  Test. — Render  10  c.c.  of  urine  strongly  alkaline 
with  potassium  hydroxide  solution  and  heat  to  boiling.  Upon 
allowing  the  heated  urine  to  stand  a  precipitate  of  phosphates, 
colored  red  by  the  contained  haematin,  is  formed.  It  is  ordi- 
narily well  to  make  a  "control"  experiment  using  normal 
urine,  before  coining  to  a  final  decision. 

Certain  substances  such  as  cascara  sagrada,  rhubarb,  san- 
tonin, and  senna  cause  the  urine  to  give  a  similar  reaction. 
Reactions  due  to  such  substances  may  be  differentiated  from 
the  true  blood  reaction  by  the  fact  that  both  the  precipitate  and 
the  pigment  of  the  former  reaction  disappear  when  treated  with 
acetic  acid,  whereas  if  the  color  is  due  to  haematin  the  acid  will 
only  dissolve  the  precipitate  of  phosphates  and  leave  the  pig- 
ment undissolved. 

2.  Teichmann's  Haemin  Test. — Place  a  small  drop  of  the 
suspected  urine  or  a  small  amount  of  the  moist  sediment  on  a 
microscopic  slide,  add  a  minute  grain  of  NaCl  and  carefully 
evaporate  to  dryness  over  a  loiv  flame.  Put  a  cover  glass  in 
place,  run  underneath  it  a  drop  of  glacial  acetic  acid  and  warm 
gently  until  the  formation  of  gas  bubbles  is  observed.  Cool 
the  preparation,  examine  under  the  microscope  and  compare 
the  form  of  the  crystals  with  those  reproduced  in  Figs.  58 
and  59,  page  164. 

3.  Heller-Teichmann  Reaction. — Produce  the  pigmented 
precipitate  according  to  directions  given  in  Heller's  test  on  p. 


URINE.  299 

298.  It'  there  Is  a  copious  precipitate  of  phosphates  and  but 
little  pigment  the  phosphates  may  be  dissolved  by  treatment 
with  acetic  acid  and  the  residue  used  in  the  formation  of  the 
haemin  crystals  according  to  directions  in  Experiment  2, p. 298. 

4.  Zeynek  and  Nencki's  Haemin  Test. — To  10  c.c.  of  the 
urine  under  examination  add  acetone  until  no  mure  precipitate 
forms.  Filter  off  the  precipitate  and  extract  it  with  10  c.c.  of 
acetone  rendered  acid  with  2-3  drops  of  hydrochloric  acid. 
Place  a  drop  of  the  resulting  colored  extract  on  a  slide,  immedi- 
ately place  a  cover  glass  in  position  and  examine  under  the 
microscope.  Compare  the  form  of  the  crystals  with  those 
shown  in  Figs.  58  and  59,  page  [64.  Haemin  crystals  pro- 
duced by  this  manipulation  are  sometimes  very  minute,  thus 
rendering-  it  difficult  to  determine  the  exact  form  of  the 
crystal. 

5.  Schalfijew's  Haemin  Test. — Place  20  c.c.  of  glacial 
acetic  acid  in  a  small  beaker  and  heat  to  8o°  C.  Add  5  c.c. 
of  the  urine  under  examination,  raise  the  temperature  to  8o° 
C.  and  stand  the  mixture  aside  to  cool.  Examine  the  crystals 
under  the  microscope  and  compare  them  with  those  shown  in 
Figs.  58  and  59,  page  164. 

6.  Guaiac  Test. — Place  5  c.c.  of  urine  in  a  test-tube  and  by 
means  of  a  pipette  introduce  a  freshly  prepared  alcoholic  solu- 
tion of  guaiac  into  the  fluid  until  a  turbidity  results;  then  add 
old  turpentine  or  hydrogen  peroxide,  drop  by  drop,  until  a  blue 
color  is  obtained.  This  is  a  very  delicate  test  when  properly 
performed.  Buckmaster  has  recently  suggested  the  use  of 
guaiaconic  acid  instead  of  the  solution  of  guaiac.  See  discus- 
sion on  page  158  and  test  on  page  163. 

7.  Spectroscopic  Examination. — Submit  the  urine  to  a 
spectroscopic  examination  according  to  the  directions  given  on 
page  169  looking  especially  for  the  absorption-bands  of  oxy- 
hemoglobin and  methaemoglobin  (see  Absorption  Spectra, 
Plate  I.). 


300  PHYSIOLOGICAL    CHEMISTRY. 

BILE. 

Both  the  pigments  and  the  acids  of  the  bile  may  be  detected 
in  the  urine  under  certain  pathological  conditions.  Of  the  pig- 
ments, bilirubin  is  the  only  one  which  has  been  positively  iden- 
tified in  fresh  urine;  the  other  pigments,  when  present,  are 
probably  derived  from  the  bilirubin.  A  urine  containing  bile 
may  be  yellowish-green  to  brown  in  color  and  when  shaken 
foams  readily.  The  staining  of  the  various  tissues  of  the 
body  through  the  absorption  of  bile  due  to  occlusion  of  the 
bile  duct  causes  a  condition  known  as  icterus  or  jaundice. 
Bile  is  always  present  in  the  urine  under  such  conditions  unless 
the  amount  of  bile  reaching  the  tissues  is  extremely  small. 

Experiments. 

Tests  for  Bile  Pigments, 
i.  Gmelin's  Test. — To  about  5  c.c.  of  concentrated  nitric 
acid  in  a  test-tube  add  an  equal  volume  of  urine  carefully  so 
that  the  two  fluids  do  not  mix.  At  the  point  of  contact  note  the 
various  colored  rings,  green,  bine,  violet,  red  and  reddish- 
yellow. 

2.  Rosenbach's  Modification  of  Gmelin's  Test. — Filter 
5  c.c.  of  urine  through  a  small  filter  paper.  Introduce  a  drop 
of  concentrated  nitric  acid  into  the  cone  of  the  paper  and 
observe  the  succession  of  colors  as  given  in  Gmelin's  test. 

3.  Huppert's  Reaction. — Thoroughly  shake  equal  volumes 
of  urine  and  milk  of  lime  in  a  test-tube.  The  pigments  unite 
with  the  calcium  and  are  precipitated.  Filter  off  the  precipi- 
tate, wash  it  with  water  and  transfer  to  a  small  beaker.  Add 
alcohol  acidified  slightly  with  hydrochloric  acid  and  warm  upon 
a  water-bath  until  the  solution  becomes  colored  an  emerald 
green. 

4.  Hammarsten's  Reaction. — To  about  5  c.c.  of  Ham- 
marsten's  reagent1  in  a  small  evaporating  dish  add  a  few  drops 

1  Hammarsten's  reagent  is  made  by  mixing  1  volume  of  25  per  cent 
nitric  acid  and  19  volumes  of  25  per  cent  hydrochloric  acid  and  then 
adding  1  volume  of  this  acid  mixture  to  4  volumes  of  95  per  cent  alcohol. 


URINE.  301 

of  urine.  A  green  color  is  produced.  If  more  of  the  reagent 
is  now  added  the  play  of  colors  as  noted  in  Gmelin's  test  may 
be  1  ibtained. 

5.  Smith's  Test. — To  2-3  c.c.  of  urine  in  a  test-tube  add 
carefully  about  5  c.c.  of  dilute  tincture  of  iodine  (1:  10)  so 
thai  the  fluids  do  not  mix.  A  green  ring  is  observed  at  the 
point  of  contact. 

Tests   for  Bile  Acids. 

1.  Pettenkofer's  Test. — To  5  c.c.  of  urine  in  a  test-tube 
add  5  drops  of  a  5  per  cent  solution  of  saccharose.  Now  in- 
cline the  tube,  run  about  2-3  c.c.  of  concentrated  sulphuric 
acid  carefully  down  the  side  and  note  the  red  ring  at  the  point 
of  contact.  Upon  slightly  agitating  the  contents  of  the  tube 
the  whole  solution  gradually  assumes  a  reddish  color.  As  the 
tube  becomes  warm,  it  should  be  cooled  in  running  water  in 
order  that  the  temperature  may  not  rise  above  700  C. 

_'.  Mylius's  Modification  of  Pettenkofer's  Test. — To  ap- 
proximately 5  c.c.  of  urine  in  a  test-tube  add  3  drops  of  a  very 
dilute  (1  :  1,000)  aqueous  solution  of  furfurol. 

HC CH 

II         II 
HC        C  •  CHO. 
\/ 
0 

Xow  incline  the  tube,  run  about  2-3  c.c.  of  concentrated  sul- 
phuric acid  carefully  down  the  side  and  note  the  red  ring  as 
above.  In  this  case  also,  upon  shaking  the  tube,  the  whole 
solution  is  colored  red.  Keep  the  temperature  below  70  C. 
as  before. 

3.  Neukomm's  Modification  of  Pettenkofer's  Test. — To 
a  few  drops  of  urine  in  an  evaporating  dish  add  a  trace  of  a 
dilute  saccharose  solution  and  one  or  more  drops  of  dilute  sul- 
phuric acid.  Evaporate  on  a  water-bath  and  observe  the 
development  of  a  violet  color  at  the  edge  of  the  evaporating 
mixture.  Discontinue  the  evaporation  as  soon  as  the  color 
is  observed. 


302  PHYSIOLOGICAL    CHEMISTRY. 

4.  v.  Udransky's  Test. — To  5  c.c.  of  urine  in  a  test-tube 
add  3-4  drops  of  a  very  dilute  (1  :  1,000)  aqueous  solution  of 
furfurol.  Place  the  thumb  over  the  top  of  the  tube  and  shake 
until  a  thick  foam  is  formed.  By  means  of  a  small  pipette 
add  2-3  drops  of  concentrated  sulphuric  acid  to  the  foam  and 
observe  the  dark  pink  coloration  produced. 

5.  Salkowski's  Test. — Render  5  c.c.  of  urine  alkaline  with 
a  few  drops  of  a  10  per  cent  sodium  carbonate  solution  and  add 
a  10  per  cent  solution  of  calcium  chloride,  drop  by  drop,  until 
the  supernatant  fluid  exhibits  the  normal  urinary  color  when 
the  contents  of  the  test-tube  are  thoroughly  mixed.  Filter  off 
the  precipitate,  and  after  washing  it,  place  it  in  a  second  tube 
with  95  per  cent  alcohol.  Acidify  the  alcohol  with  hydro- 
chloric acid  and,  if  necessary,  shake  the  tube  to  bring  the  pre- 
cipitate into  solution.  Heat  the  solution  to  boiling  and  observe 
the  appearance  of  a  green  color  which  changes  through  blue 
and  violet  to  red;  if  no  bile  is  present  the  solution  does  not 
undergo  any  color  change.  This  test  will  frequently  exhibit 
greater  delicacy  that  Gmelin's  test. 

6.  Hay's  Test. — Cool  about  10  c.c.  of  urine  in  a  test-tube 
to  1 70  C.  or  lower,  and  sprinkle  a  little  finely  pulverized  sul- 
phur upon  the  surface  of  the  fluid.  The  presence  of  bile  acids 
is  indicated  if  the  sulphur  sinks  to  the  bottom  of  the  liquid,  the 
rapidity  with  which  the  sulphur  sinks  depending  upon  the 
amount  of  bile  acids  present  in  the  urine.  The  test  is  said  to 
react  with  bile  acids  when  the  latter  are  present  in  the  propor- 
tion 1  :  120,000. 

Some  investigators  claim  that  it  is  impossible  to  differentiate 
between  bile  acids  and  bile  pigments  by  this  test. 

CH3 
I 

ACETONE,    C  =  0. 

I 

CH3 

It  was  formerly  very  generally  believed  that  acetone  appeared 


URINE.  303 

in  the  urine  under  pathological  conditions  because  of  increased 
proteid  decomposition.  It  is  now  generally  thought  that,  in 
man,  the  output  of  acetone  arises  principally  from  the  breaking 
down  of  fatty  tissues  or  fatty  foods  within  the  organism. 
The  quantity  of  acetone  eliminated  has  been  shown  to  increase 
when  the  subject  is  fed  an  abundance  of  fat-containing  food 
as  well  as  during  fasting,  whereas  a  replacement  of  the  fat 
with  carbohydrates  is  followed  by  a  marked  decrease  in  the 
acetone  excretion.  Conditions  are  different  with  certain  of 
the  lower  animals.  With  the  dog,  for  instance,  the  output 
of  acetone  is  not  diminished  when  the  animal  is  fed  upon  a 
carbohydrate  diet,  is  decreased  during  fasting  and  increased 
when  the  animal  is  fed  upon  a  diet  of  meat. 

Acetone  and  the  closely  related  bodies,  /?-oxybutyric  acid  and 
diacetic  acid,  are  generally  classified  as  the  acetone  bodies. 
They  are  all  associated  with  a  deranged  metabolic  function  and 
may  appear  in  the  urine  together  or  separately,  depending  upon 
the  conditions.  Acetone  and  diacetic  acid  may  occur  alone  in 
the  urine  but  /8-oxybutyric  acid  is  never  found  except  in  con- 
junction with  one  or  the  other  of  these  bodies.  Acetone  and 
diacetic  acid  arise  chiefly  from  the  oxidation  of  /?-oxybutyric 
acid.  The  relation  existing  between  these  three  bodies  is 
shown  in  the  following  reactions  : 

(a)  CH3-CH(OH)-CH2-COOH  +  0  = 

0  -oxvbutyric  acid. 

CH3CO  •  CH2  •  COOH  +  H20. 

Diacetic  acid. 

(b)  CH3CO-CH2-COOH=(CH3)2CO  +  C02. 

Diacetic  acid.  Acetone. 

Acetone,  chemically  considered,  is  a  ketone,  di-m ethyl  ketone. 
When  pure  it  is  a  liquid  which  possesses  a  characteristic  aro- 
matic fruit-like  odor,  boils  at  56— 570  C.  and  is  miscible  with 
water,  alcohol  or  ether  in  all  proportions.  Acetone  is  a 
physiological  as  well  as  a  pathological  constituent  of  the  urine 
and  under  normal  conditions  the  daily  output  is  about  0.0 1- 
0.03  gram. 


3O4  PHYSIOLOGICAL    CHEMISTRY. 

Pathologically,  the  elimination  of  acetone  is  often  greatly 
increased  and  at  such  times  a  condition  of  acetonuria  is  said  to 
exist.  This  pathological  acetonuria  may  accompany  diabetes 
mellitus,  scarlet  fever,  typhoid  fever,  pneumonia,  nephritis, 
phosphorus  poisoning,  grave  anaemias,  fasting'  and  a  deranged 
digestive  function;  it  also  frequently  accompanies  auto-intoxi- 
cation and  chloroform  anaesthesia.  The  types  of  acetonuria 
most  frequently  met  with  are  those  noted  in  febrile  conditions 
and  in  advanced  cases  of  diabetes  mellitus. 

Experiments. 

1.  Isolation  from  the  Urine. — In  order  to  facilitate  the  de- 
tection of  acetone  in  the  urine,  the  specimen  under  examination 
should  be  distilled  and  the  tests  as  given  below  applied  to  the 
resulting  distillate.  If  it  is  not  convenient  to  distil  the  urine, 
the  tests  may  be  conducted  upon  the  undistilled  fluid.  To 
obtain  an  acetone  distillate  proceed  as  follows :  Place  100-250 
c.c.  of  urine  in  a  distillation  flask  or  retort  and  render  it  acid 
with  acetic  acid.  Collect  about  one-third  of  the  original 
volume  of  fluid  as  a  distillate,  add  5  drops  of  10  per  cent  hydro- 
chloric acid  and  redistil  about  one-half  of  this  volume.  With 
this  final  distillate  conduct  the  tests  as  given  below. 

2.  Gunning's  Iodoform  Test. — To  about  5  c.c.  of  the  urine 
or  distillate  in  a  test-tube  add  a  few  drops  of  Lugol's  solution1 
or  ordinary  iodine  solution  (I  in  KI)  and  enough  NH4OH  to 
form  a  black  precipitate  (nitrogen  iodide).  Allow  the  tube  to 
stand  (the  length  of  time  depending  upon  the  content  of  acetone 
in  the  fluid  under  examination)  and  note  the  formation  of  a 
yellowish  sediment  consisting  of  iodoform.  Examine  the  sedi- 
ment under  the  microscope  and  compare  the  form  of  the  crys- 
tals with  those  shown  in  Fig.  6,  p.  21.  If  the  crystals  are  not 
well  formed  recrystallize  them  from  ether  and  examine  again. 
The  crystals  of  iodoform  should  not  be  confounded  with  those 
of  stellar  phosphate  (Fig.  j6,  p.  193)  which  may  be  formed  in 

'Lugol's  solution  may  be  prepared  by  dissolving  5  grams  of  iodine  and 
10  grams  of  potassium  iodide  in  100  c.c.  of  distilled  water. 


trim:.  305 

this  test,  particularly  if  made  upon  the  undistilled  urine.  This 
test  is  preferable  to  Lieben's  test  (4)  since  no  substance  other 
than  acetone  will  produce  iodoform  when  treated  accord- 
ing to  the  directions  for  this  test;  both  alcohol  and  aldehyde 
yield  iodoform  when  tested  by  Lieben's  test. 

Gunning's  test  is  rather  the  most  satisfactory  test  yet  sug- 
gested for  the  detection  of  acetone,  and  may  be  used  with  good 
results  even  upon  the  undistilled  urine.  In  some  instances 
where  the  amount  of  acetone  present  is  very  small  it  is  neces- 
sary to  allow  the  tube  to  stand  24  hours  before  making  the 
examination  for  iodoform  crystals.  This  test  serves  to  detect 
acetone  when  present  in  the  ratio  1  :  100,000. 

3.  Legal's  Test. — Introduce  about  5  c.c.  of  the  urine  or 
distillate  into  a  test-tube,  add  a  few  drops  of  a  freshly  prepared 
aqueous  solution  of  sodium  nitro-prusside  and  render  the  mix- 
ture alkaline  with  potassium  hydroxide.  A  ruby  red  color, 
clue  to  creatinin,  a  normal  urinary  constituent,  is  produced  (see 
Weyl's  test.  p.  2^2).  Add  an  excess  of  acetic  acid  and  if  acetone 
is  present  the  red  color  will  be  intensified,  whereas  in  the  ab- 
sence of  acetone  a  yellow  color  will  result.  Make  a  control 
test  upon  normal  urine  to  show  that  this  is  so.  A  similar  red 
color  may  be  produced  by  paracresol  in  urines  containing  no 
acetone. 

4.  Lieben's  Test. — Introduce  5  c.c.  of  the  urine  or  distillate 
into  a  test-tube,  render  it  alkaline  with  potassium  hydroxide  and 
add  1-2  c.c.  of  iodine  solution,  drop  by  drop.  If  acetone  is 
present  a  yellowish  precipitate  of  iodoform  will  be  produced. 
Identifv  the  iodoform  by  means  of  its  characteristic  odor  and 
its  typical  crystalline  form  (see  Fig.  6.  p.  21).  While  fully 
as  delicate  as  Gunning's  test  (2)  this  test  is  not  as  accurate, 
since  by  means  of  the  procedure  involved,  either  alcohol  or 
aldehyde  will  yield  a  precipitate  of  iodoform.  This  test  is 
especially  liable  to  lead  to  erroneous  deductions  when  urines 
from  the  advanced  stages  of  diabetes  are  under  examination, 
because  of  the  presence  of  alcohol  formed  from  the  sugar 
through  fermentative  processes. 


306  PHYSIOLOGICAL    CHEMISTRY. 

5.  Reynolds-Gunning  Test. — This  test  depends  upon  the 
solubility  of  mercuric  oxide  in  acetone  and  is  performed  as  fol- 
lows :  To  5  c.c.  of  the  urine  or  distillate  add  a  few  drops  of 
mercuric  chloride,  render  the  solution  alkaline  with  potassium 
hydroxide  and  add  an  equal  volume  of  95  per  cent  alcohol. 
Shake  thoroughly  in  order  to  bring  the  major  portion  of  the 
mercuric  oxide  into  solution  and  filter.  Render  the  clear 
filtrate  faintly  acid  with  hydrochloric  acid  and  stratify  some 
ammonium  sulphide,  (NH4)2S,  upon  this  acid  solution.  At 
the  zone  of  contact  a  blackish-gray  ring  of  precipitated  mercu- 
ric sulphide,  HgS,  will  form.  Aldehyde  also  responds  to  this 
test.  Aldehyde,  however,  has  never  been  detected  in  the  urine 
and  could  only  be  present  in  this  instance  if  the  acidified  urine 

was  distilled  too  far. 

CH3 
I 

DIACETIC  ACID,  C  =  0 

CHo-COOH. 

Diacetic  or  acetoacetic  acid  occurs  in  the  urine  only  under 
pathological  conditions  and  is  rarely  found  except  associated 
with  acetone.  It  is  formed  from  /?-oxybutyric  acid,  another  of 
the  acetone  bodies,  and  upon  decomposition  yields  acetone  and 
carbon  dioxide.  Diaceturia  occurs  ordinarily  under  the  same 
conditions  as  the  pathological  acetonuria,  i.  e.,  in  fevers,  dia- 
betes, etc.  (see  p.  304).  If  very  little  diacetic  acid  is  formed 
it  may  all  be  transformed  into  acetone,  whereas  if  a  larger 
quantity  is  produced  both  acetone  and  diacetic  acid  may  be 
present  in  the  urine.  Diaceturia  is  most  frequently  observed 
in  children,  especially  accompanying  fevers  and  digestive  dis- 
orders; it  is  perhaps  less  frequently  observed  in  adults,  but 
when  present,  particularly  in  fevers  and  diabetes,  it  is  fre- 
quently followed  by  fatal  coma. 

Diacetic  acid  is  a  colorless  liquid  which  is  miscible  with 
water,  alcohol,  and  ether,  in  all  proportions.  It  differs  from 
acetone  in  giving  a  violet-red  or  Bordeaux-red  color  with  a 
dilute  solution  of  ferric  chloride. 


URINE.  307 

Experiments. 
1.  Gerhardt's  Test. — To  5  cc  of  urine  in  a  test-tube  add 

ferric  chloride  solution,  drop  by  drop,  until  no  more  precipi- 
tate forms.  In  the  presence  of  diacetic  acid  a  Bordeaux-red 
color  is  produced ;  this  color  may  be  somewhat  masked  by  the 
precipitate  of  ferric  phosphate,  in  which  case  the  fluid  should 
be  filtered. 

A  positive  result  from  the  above*  manipulation  simply  indi- 
cates the  possible  presence  of  diacetic  acid.  Before  making  a 
final  decision  regarding  the  presence  of  this  body  make  the  two 
following  control  experiments: 

1  (/ )  Place  5  cc.  of  urine  in  a  test-tube  and  boil  it  vigorously 
for  3-5  minutes.  Cool  the  tube  and,  with  the  boiled  urine, 
make  the  test  as  given  above.  As  has  been  already  stated, 
diacetic  acid  yields  acetone  upon  decomposition  and  acetone 
does  not  give  a  Bordeaux-red  color  with  ferric  chloride.  By 
boiling  as  indicated  above  therefore,  any  diacetic  acid  present 
would  be  decomposed  into  acetone  and  carbon  dioxide  and  the 
test  upon  the  resulting  fluid  would  be  negative.  If  positive 
the  color  is  due  to  the  presence  of  bodies  other  than  diacetic 
acid. 

I  />)  Place  5  cc.  of  urine  in  a  test-tube,  acidify  with  H2S04, 
tc  free  diacetic  acid  from  its  salts,  and  carefully  extract  the 
mixture  with  ether  by  shaking.  If  diacetic  acid  is  present  it 
will  be  extracted  by  the  ether.  Now  remove  the  ethereal  solu- 
tion and  add  to  it  an  equal  volume  of  dilute  ferric  chloride; 
diacetic  acid  is  indicated  by  the  production  of  the  character- 
istic Bordeaux-red  color.  This  color  disappears  spontaneously 
in  24-48  hours.  Such  substances  as  antipyrin,  kairin,  phena- 
cetin.  salicylic  acid,  salicylates,  sodium  acetate,  sulphocyanides 
and  thallin  yield  a  similar  red  color  under  these  conditions,  but 
when  due  to  the  presence  of  any  of  these  substances  the  color 
does  not  disappear  spontaneously  but  may  remain  permanent 
for  days.  Many  of  these  disturbing  substances  are  soluble  in 
benzene  or  chloroform  and  may  be  removed  from  the  urine  by 
this  means  before  extracting  with  ether  as  above.  Diacetic 
acid  is  insoluble  in  benzene  or  chloroform. 


3CS  PHYSIOLOGICAL    CHEMISTRY. 

2.  Arnold-Lipliawsky  Reaction. — This  reaction  is  some- 
what more  delicate  than  Gerhardt's  test  (i)  and  serves  to  de- 
tect diacetic  acid  when  present  in  the  proportion  i :  25,000.  It 
is  also  negative  toward  acetone,  /8-oxybutyric  acid  and  the  inter- 
fering drugs  mentioned  as  causing'  erroneous  deductions  in  the 
application  of  Gerhardt's  test.  If  the  urine  under  examination 
is  highly  pigmented  it  should  be  partly  decolorized  by  means 
of  animal  charcoal  before  applying  the  test  as  indicated  below. 

Place  5  c.c.  of  the  urine  under  examination  and  an  equal 
volume  of  the  Arnold-Lipliawsky  reagent1  in  a  test-tube,  add  a 
few  drops  of  concentrated  ammonia  and  shake  the  tube  vigor- 
ously. Note  the  production  of  a  brick-red  color.  Take  1-2 
c.c.  of  this  colored  solution,  add  10-20  c.c.  of  hydrochloric 
acid  (sp.  gr.  1.19),  3  c.c.  of  chloroform  and  2-4  drops  of 
ferric  chloride  solution  and  carefully  mix  the  fluids  without 
shaking.  Diacetic  acid  is  indicated  by  the  chloroform  assum- 
ing a  violet  or  blue  color;  if  diacetic  acid  is  absent  the  color 
may  be  yellow  or  light  red. 

H    OH  H 

I       I        I 

/3-OXYBUTYRIC  ACID,  H  —  C  —  C  —  0  — COOH. 

I    I     I 

H    H     H 

This  acid  does  not  occur  as  a  normal  constituent  of  urine 
but  is  found  only  under  pathological  conditions  and  then 
always  in  conjunction  with  either  acetone  or  diacetic  acid. 
Either  of  these  bodies  may  be  formed  from  /3-oxybutyric  acid 
under   proper   conditions.     It   is   present   in   especially   large 

1  This  reagent  consists  of  two  definite  solutions  which  are  ordinarily 
preserved  separately  and  mixed  just  before  using.  The  two  solutions  are 
prepared  as  follows : 

(a)  One  per  cent  aqueous  solution  of  potassium  nitrite. 

(b)  One  gram  of  />-amino-acetophenon  dissolved  in  100  c.c.  of  distilled 
water  and  enough  hydrochloric  acid  (about  2  c.c.)  added,  drop  by  drop, 
to  cause  the  solution,  which  is  at  first  yellow,  to  become  entirely  colorless. 
An  excess  of  acid  must  be  avoided. 

Before  using,  a  and  b  are  mixed  in  the  ratio  1 :  2. 


URINE.  3°9 

amount  in  severe  cases  of  diabetes  and  has  also  been  detected 
in  digestive  disturbances,  continued  fevers,  scurvy,  measles  and 
in  starvation.     It  is  probable  that,  in  man.  |8-oxybutyric  acid, 

in  common  with  acetone  and  diacetic  acid,  arises  principally 
from  the  breaking  down  of  fatty  tissues  within  the  organism. 
The  condition  in  which  large  amounts  of  acetone  and  diacetic 
acid,  and  in  severe  cases  |8-oxybutyric  acid  also,  are  excreted 
in  the  urine  is  known  as  "  acidosis."  In  diabetes  the  deranged 
metabolic  conditions  cause  the  production  of  great  quantities 
of  these  substances  which  lead  to  an  acid  intoxication  and 
ultimately  to  diabetic  coma. 

Ordinarily  /B-oxybutyric  acid  is  an  odorless,  transparent 
syrup,  which  is  laevorotatory  and  easily  soluble  in  water,  alco- 
hol and  ether;  it  may  be  obtained  in  crystalline  form. 

Experiments. 

i.  Polariscopic  Examination. — Subject  some  of  the  urine 
(free  from  proteid)  to  the  ordinary  fermentation  test  (see 
page  288).  This  will  remove  dextrose  and  lsevulose,  which 
would  interfere  with  the  polariscopic  test.  Now  examine  the 
fermented  fluid  in  the  polariscope  and  if  it  is  laevorotatory 
the  presence  of  0-oxybutyric  acid  is  indicated.  This  test  is  not 
absolutely  reliable,  however,  since  conjugate  glycuronates  are 
also  laevorotatory  after  fermentation. 

2.  Kiilz's  Test. — Evaporate  the  urine,  after  fermenting 
it  as  indicated  in  the  last  test,  to  a  syrup,  add  an  equal  volume 
of  concentrated  sulphuric  acid  and  distil  the  mixture  directly 
without  cooling.  Under  these  conditions  a-crotonic  acid  is 
formed  and  is  present  in  the  distillate.  Allow  the  distillate  to 
cool  slowly  and  note  the  formation  of  crystals  of  a-crotonic  acid 
which  are  soluble  in  ether  and  melt  at  72  °  C.  In  case  very 
slight  traces  of  /?-oxybutyric  acid  be  present  in  the  urine 
under  examination  the  amount  of  a-crotonic  acid  formed  may 
be  too  small  to  yield  a  crystalline  product.  In  this  event  the 
distillate  should  be  extracted  with  ether,  the  ethereal  extract 
evaporated  and  the  residue  washed  with  water.     Under  these 


3IO  PHYSIOLOCxICAL    CHEMISTRY. 

conditions  the  impurities  will  be  removed  and  the  a-crotonic 
acid  will  remain  behind  as  a  residue.  The  melting-point  of 
this  residue  may  then  be  determined. 


CONJUGATE  GLYCURONATES. 

Glycuronic  acid  does  not  occur  free  in  the  urine  but  is 
found,  for  the  most  part,  in  combination  with  phenol.  Much 
smaller  quantities  are  excreted  in  combination  with  indoxyl 
and  skatoxyl.  The  total  content  of  conjugate  glycuronates 
seldom  exceeds  0.004  per  cent  under  normal  conditions.  The 
output  may  be  very  greatly  increased  as  the  result  of  the 
administration  of  antipyrin,  borneol,  camphor,  chloral,  men- 
thol, morphine,  naphthol,  turpentine,  etc.  The  glycuronates 
as  a  group  are  lsevorotatory,  whereas  glycuronic  acid  is 
dextro-rotatory.  Most  of  the  glycuronates  reduce  alkaline 
metallic  oxides  and  so  introduce  an  error  in  the  examination 
of  urine  for  sugar.  Conjugate  glycuronates  often  occur 
associated  with  dextrose  in  glycosuria,  diabetes  mellitus  and 
in  some  other  disorders.  As  a  class  the  glycuronates  are  non- 
fermentable. 

Experiments. 

1.  Fermentation-Reduction  Test.  —  Test  the  urine  by 
Fehling's  test.  If  there  is  reduction  try  Barfoed's  test.  If 
negative  this  indicates  the  absence  of  dextrose.  A  negative 
fermentation  test  would  now  indicate  the  presence  of  conju- 
gate glycuronates  (or  lactose  in  rare  cases). 

If  dextrose  is  present  in  the  urine  tested  for  glycuronates 
the  urine  must  first  be  subjected  to  a  polariscopic  examina- 
tion, then  fermented  and  a  second  polariscopic  examination 
made.  The  sugar  being  dextro-rotatory  and  fermentable 
and  the  glycuronates  being  laevorotatory  and  non- fermentable 
the  second  polariscopic  test  will  show  a  lsevorotation  indica- 
tive of  conjugate  glycuronates. 

2.  Tollens'  Reaction. — Make  this  test  according  to  direct- 
ions given  under  Pentoses,  page  311. 


URINE.  311 

PENTOSES. 

We  have  two  distinct  types  of  pentosuria,  i.  c,  alimentary 
pentosuria,  resulting  from  the  ingestion  of  large  quantities 
of  pentose-rich  vegetables  such  as  prunes,  cherries,  grapes  or 
plums,  and  fruit  juices,  in  which  condition  the  pentoses  appear 
only  temporarily  in  the  urine;  and  the  chrome  form  of  pento- 
suria, in  which  the  output  of  pentoses  bears  no  relation  what- 
ever to  the  quantity  and  nature  of  the  pentose  content  of 
the  food  eaten.  In  occurring  in  these  two  forms,  pentosuria 
resembles  glycosuria  (see  page  283),  but  it  is  definitely  known 
that  pentosuria  bears  no  relation  to  diabetes  mellitus  and 
there  is  no  generally  accepted  theory  to  account  for  the  occur- 
rence of  the  chronic  form  of  pentosuria.  The  pentose  de- 
tected most  frequently  in  the  urine  is  arabinose,  the  inactive 
form  generally  occurring  in  chronic  pentosuria  and  the  lsevo- 
rotatory  variety  occurring  in  the  alimentary  type  of  the 
disorder. 

Experiments. 

1.  Tollens'  Reaction. — To  equal  volumes  of  urine  and 
hydrochloric  acid  (sp.  gr.  1.09)  add  a  little  phloroglucin  and 
heat  the  mixture  on  a  boiling  water-bath.  Pentose,  galactose, 
laevulose  or  glycuronic  acid  will  be  indicated  by  the  appearance 
of  a  red  color.  To  differentiate  between  these  bodies  examine 
by  the  spectroscope  and  look  for  the  absorption  band  between  D 
and  E  given  by  pentoses  and  glycuronic  acid,  and  then  differ- 
entiate between  the  two  latter  bodies  by  the  melting-points  of 
their  osazons. 

2.  Orcin  Test. — Place  equal  volumes  of  urine  and  hydro- 
chloric acid  (sp.  gr.  1.09)  in  a  test-tube,  add  a  small  amount 
of  orcin,  and  heat  the  mixture  to  boiling.  Color  changes 
from  red,  through  reddish-blue  to  green  will  be  noted.  When 
the  solution  becomes  green  it  should  be  shaken  in  a  separatory 
funnel  with  a  little  amyl  alcohol,  and  the  alcoholic  extract 
examined  spectroscopically.  An  absorption  band  between  C 
and  D  will  be  observed. 


312  THYSIOLOGICAL    CHEMISTRY. 

FAT. 

When  fat  finds  its  way  into  the  urine  through  a  lesion 
which  brings  some  portion  of  the  urinary  passages  into  com- 
munication with  the  lymphatic  system  a  condition  known  as 
chyhtria  is  established.  The  turbid  or  milky  appearance  of 
such  urine  is  due  to  its  content  of  chyle.  This  disease  is 
encountered  most  frequently  in  tropical  countries,  but  is  not 
entirely  unknown  in  more  temperate  climates.  Albumin  is  a 
constant  constituent  of  the  urine  in  chyluria.  Upon  shaking 
a  chylous  urine  with  ether  the  fat  is  dissolved  by  the  ether 
and  the  urine  becomes  clearer  or  entirely  clear. 

HiEMATOPORPHYRIN. 

Urine  containing  this  body  is  occasionally  met  with  in 
various  diseases  but  more  frequently  after  the  use  of  quinine, 
tetronal,  trional  and  especially  sulphonal.  Such  urines  ordi- 
narily possess  a  reddish  tint,  the  depth  of  color  varying  greatly 
under  different  conditions. 

Experiments. 

i.  Spectroscopic  Examination. — To  ioo  c.c.  of  urine 
add  about  20  c.c.  of  a  10  per  cent  solution  of  KOH  or 
NH4OH.  The  precipitate  which  forms  consists  principally 
of  earthy  phosphates  to  which  the  haematoporphyrin  adheres 
and  is  carried  down.  Filter  off  the  precipitate,  wash  it  and 
transfer  to  a  flask  and  warm  with  alcohol  acidified  with  hy- 
drochloric acid.  By  this  process  the  haematoporphyrin  is  dis- 
solved and  on  filtering  will  be  found  in  the  filtrate  and  may 
be  identified  by  means  of  the  spectroscope  (see  page  173,  and 
Absorption  Spectra,  Plate  II). 

2.  Acetic  Acid  Test. — To  100  c.c.  of  urine  add  5  c.c.  of 
glacial  acetic  acid  and  allow  the  mixture  to  stand  48  hours. 
Haematoporphyrin  deposits  in  the  form  of  a  precipitate. 


URINE.  313 

LACTOSE. 

Lactose  is  rarely  found  in  the  urine  except  as  it  is  excreted 
by  women  during  pregnancy,  during  the  nursing  period  or 
soon  after  weaning.  It  is  rather  difficult  t«>  show  the  pres- 
ence of  lactose  in  the  urine  in  a  satisfactory  manner,  since 
the  formation  of  the  characteristic  lactosazon  is  not  attended 
with  any  great  measure  of  success  under  these  conditions. 
It  is,  however,  comparatively  easy  to  show  that  it  is  not  dex- 
trose, for.  while  it  responds  to  reduction  tests,  it  docs  not 
ferment  with  pure  yeast  and  does  not  give  a  dextrosazon. 
An  absolutely  conclusive  test,  of  course,  is  the  isolation  of 
the  lactose  in  crystalline  form  (Fig.  75,  p.  189)  from  the  urine. 

Experiments. 

1.  Rubner's  Test. — To  10  c.c.  of  urine  in  a  small  beaker 
add  some  plumbic  acetate,  in  substance,  heat  to  boiling  and 
add  XH4OH  until  no  more  precipitate  is  dissolved.  In  the 
presence  of  lactose  a  brick-red  or  rose-red  color  develops, 
whereas  dextrose  gives  a  coffee-brown  color,  maltose  a  light 
yellow  color  and  lsevulose  no  color  at  all  under  the  same 
conditions. 

2.  Compound  Test. — Try  the  phenylhydrazin  test,  the 
fermentation  test  and  Barfoed's  test  according  to  directions 
given  under  Dextrose,  pages  283,  288  and  289.  If  these  are 
negative,  try  Xylander's  test,  page  288.  If  this  last  test  is 
positive,  the  presence  of  lactose  is  indicated. 

L^VULOSE. 

Diabetic  urine  frequently  possesses  the  power  of  rotating 
the  plane  of  polarized  light  to  the  left,  thus  indicating  the 
presence  of  a  l?evorotatory  substance.  This  kevorotation  is 
sometimes  due  to  the  presence  of  laevulose,  although  not  nec- 
essarily confined  to  this  carbohydrate,  since  conjugate  glycu- 
ronates  and  /?-oxybutyric  acid,  two  other  lsevorotatory  bodies, 


314  PHYSIOLOGICAL    CHEMISTRY. 

are  frequently  found  in  the  urine  of  diabetics.  Laevulose  is 
invariably  accompanied  by  dextrose  in  diabetic  urine,  but 
Icevulo&uria  has  been  observed  as  a  separate  anomaly.  The 
presence  of  laevulose  may  be  inferred  when  the  percentage 
of  sugar,  as  determined  by  the  titration  method,  is  greater 
than  the  percentage  indicated  by  the  polariscopic  examination. 

Experiments. 

1.  Seliwanoff's  Reaction. — If  a  solution  of  resorcin  in 
dilute  HC1  (1  volume  of  concentrated  HC1  to  2  volumes  of 
H20)  be  warmed  with  an  equal  volume  of  a  urine  containing 
laevulose,  the  liquid  will  become  red  and  a  precipitate  will 
separate.  The  precipitate  may  be  dissolved  in  alcohol  to  which 
it  will  impart  a  striking  red  color. 

2.  Phenylhydrazin  Test. — Make  the  test  according  to 
directions  under  Dextrose,  1  page  283. 

3.  Polariscopic  Examination. — A  simple  polariscopic  ex- 
amination, when  taken  in  connection  with  other  ordinary  tests, 
will  furnish  the  requisite  data  regarding  the  presence  of 
laevulose,  provided  laevulose  is  not  accompanied  by  other  laevo- 
rotatory  substances,  such  as  conjugate  glycuronates  and 
/?-oxybutyric  acid. 

INOSIT. 

Inosit  occasionally  occurs  in  the  urine  in  albuminuria, 
diabetes  mellitus  and  diabetes  insipidus.  It  is  claimed  also 
that  copious  water-drinking  causes  this  body  to  appear  in  the 
urine.  By  some  investigators  inosit  is  believed  to  occur  in 
traces  in  normal  urine. 

Experiment. 
1.  Detection  of  Inosit. — Acidify  the  urine  with  concen- 
trated nitric  acid  and  evaporate  nearly  to  dryness.  Add  a 
few  drops  of  NH4OH  and  a  little  CaCl2  solution  to  the  moist 
residue  and  evaporate  the  mixture  to  dryness.  In  the  pres- 
ence of  inosit  (0.001  gram)  a  bright  red  color  is  obtained. 


URINE.  315 


LAIOSE. 


This  substance  is  occasionally  found  in  the  urine  in  severe 
cases  of  diabetes  mellitus.  By  some  investigators  laiose  is 
classed  with  the  sugars.  It  resembles  laevulose  in  that  it  has 
the  property  of  reducing  certain  metallic  oxides  and  is  laevoro- 
tatbry,  but  differs  from  laevulose  in  being  amorphous,  non- 
fermentable  "and  in  not  possessing  a  sweet  taste. 

MELANINS. 

These  pigments  never  occur  normally  in  the  urine  but  are 
present  under  certain  pathological  conditions,  their  presence 
being  especially  associated  with  melanotic  tumors.  Ordi- 
narily the  freshly  passed  urine  is  clear,  but  upon  exposure  to 
the  air  the  color  deepens  and  may  at  the  last  be  very  dark 
brown  or  black  in  color.  The  pigment  is  probably  present  in 
the  form  of  a  chromogen  or  melanogen  and  upon  coming  in 
contact  with  the  air  oxidation  occurs,  causing  the  transfor- 
mation of  the  melanogen  into  melanin  and  consequently  the 
darkening  of  the  urine. 

It  is  claimed  that  melanuria  is  proof  of  the  formation  of  a 
visceral  melanotic  growth.  In  many  instances,  without  doubt, 
urines  rich  in  indican  have  been  wrongly  taken  as  diagnostic 
proof  of  melanuria.  The  pigment  melanin  is  sometimes  mis- 
taken for  indigo  and  melanogen  for  indican.  It  is  compara- 
tively easy  to  differentiate  between  indigo  and  melanin 
through  the  solubility  of  the  former  in  chloroform. 

In  rare  cases  melanin  is  found  in  urinary  sediment  in  the 
form  of  fine  amorphous  granules. 

Experiments. 
1.  Zeller's  Test. — To  50  c.c.  of  urine  in  a  small  beaker 
add  an  equal  volume  of  bromine  water.     In  the  presence  of 
melanin   a  yellow   precipitate  will    form   and   will   gradually 
darken  in  color,  ultimately  becoming  black. 


6 


l6  PHYSIOLOGICAL    CHEMISTRY. 


2.  von  Jaksch-Pollak  Reaction. — Add  a  few  drops  of 
ferric  chloride  solution  to  10  c.c.  of  urine  in  a  test-tube  and  note 
the  formation  of  a  gray  color.  Upon  the  further  addition  of 
the  chloride  a  dark  precipitate  forms,  consisting  of  phos- 
phates and  adhering  melanin.  An  excess  of  ferric  chloride 
causes  the  precipitate  to  dissolve. 

This  is  the  most  satisfactory  test  for  the  identification  of 
melanin  in  the  urine. 

UROROSEIN. 

This  is  a  pigment  which  is  not  present  in  normal  urine  but 
may  be  detected  in  the  urine  of  various  diseases,  such  as  pul- 
monary tuberculosis,  typhoid  fever,  nephritis  and  stomach  dis- 
orders. Urorosein,  in  common  with  various  other  pigments, 
does  not  occur  preformed  in  the  urine,  but  is  present  in  the 
form  of  a  chromogen,  which  is  transformed  into  the  pigment 
upon  treatment  with  a  mineral  acid. 

Experiments. 

i.  Robin's  Reaction. — Acidify  10  c.c.  of  urine  with 
about  15  drops  of  concentrated  hydrochloric  acid.  Upon 
allowing  the  acidified  urine  to  stand,  a  rose-red  color  will 
appear  if  urorosein  is  present. 

2.  Nencki  and  Sieber's  Reaction. — To  100  c.c.  of  urine  in 
a  beaker  add  10  c.c.  of  25  per  cent  sulphuric  acid.  Allow  the 
acidified  urine  to  stand  and  note  the  appearance  of  a  rose-red 
color.  The  pigment  may  be  separated  by  extraction  with 
amyl  alcohol. 

UNKNOWN    SUBSTANCES. 

Ehrlich's  Diazo  Reaction. — Place  equal  volumes  of  urine 

and  Ehrlich's  diazobenzenesulphonic  acid  reagent1   in  a  test- 

1  Two  separate  solutions  should  be  prepared  and  mixed  in  definite  pro- 
portions when  needed  for  use. 

(a)  Five  grams  of  sodium  nitrite  dissolved  in   1   liter  of  ditilled  water. 

(b)  Five  grams  of  sulphanilic  acid  and  50  c.c.  of  hydrochloric  acid  in  1 
liter  of  distilled  water. 


URINE.  317 

tube,  mix  thoroughly  by  shaking  and  quickly  add  ammonium 
hydroxide  in  excess.  The  tesl  is  positive  if  both  the  fluid  and 
the  foam  assume  a  red  color.  If  the  tube  is  allowed  to  stand 
a  precipitate  fi  irms,  the  upper  p< >rtii >n  1  >f  which  exhibits  a  blue, 
green,  greenish-black  or  violet  color.  Normal  urine  gives  a 
brownish-yellow    reaction  with  the  above  manipulation. 

The  exact  nature  of  the  substance  or  substances  upon  whose 
presence  in  the  urine  this  reaction  depends  is  not  well  under- 
stood. Some  investigators  claim  that  a  positive  reaction  in- 
dicates an  abnormal  decomposition  of  proteid  material, 
whereas  others  assume  it  to  be  due  to  an  increased  excretion 
of  alloxyproteic  acid,  oxyproteic  acid  or  uroferric  acid. 

The  reaction  may  be  taken  as  a  metabolic  symptom  of 
certain  disorders,  which  is  of  value  diagnostically  only  when 
taken  in  connection  with  the  other  symptoms.  The  reaction 
appears  principally  in  the  urine  in  febrile  disorders  and  in 
particular  in  the  urine  in  typhoid  fever,  tuberculosis  and 
measles.  The  reaction  has  also  been  obtained  in  the  urine  in 
various  other  disorders  such  as  carcinoma,  chronic  rheuma- 
tism, diphtheria,  erysipelas,  pleurisy,  pneumonia,  scarlet  fever, 
syphilis,  typhus,  etc.  The  administration  of  alcohol,  chrysa- 
robin.  creosote,  cresol,  dionin,  guaiacol,  heroin,  morphine, 
naphthalene,  opium,  phenol,  tannic  acid,  etc.,  will  also  cause 
the  urine  to  give  a  positive  reaction. 

The  following  chemical  reactions  take  place  in  this  test : 

(a)  NaN02  +  HCl  =  HN02  +  NaCl. 

NH2  N 

/  /       \ 

(b)  CCH4  +HNOo  =  C0H4  N  +  2PL0. 

\  \       / 

HSO3  S03 

Sulphanilic   acid.  Diazo-benzenesulphonic   acid. 

Solutions  a  and  b  should  be  preserved  in  well  stoppered  vessels  and 
mixed  in  the  proportion  1  :  50  when  required.  Green  asserts  that  greater 
delicacy  is  secured  by  mixing  the  solutions  in  the  proportion  1  :  100.  The 
sodium  nitrite  deteriorates  upon  standing  and  becomes  unfit  for  use  in  the 
course  of  a  few  weeks. 


CHAPTER    XIX. 


URINE:   ORGANIZED   AND   UNORGANIZED 
SEDIMENTS. 

The  data  obtained  from  carefully  conducted  microscopical 
examinations  of  the  sediment  of  certain  pathological  urines 
are  of  very  great  importance,  diagnostically.  Too  little  em- 
phasis is  sometimes  placed  upon  the  value  of  such  findings. 


Fig.  97. 


Fig.  98. 


The   Purdy   Electric   Centrifuge. 


Sediment   Tube   for  the   Purdy 
Electric  Centrifuge. 


The  sedimentary  constituents  may  be  divided  into  two 
classes,  i.  e.,  organized  and  unorganized.  The  sediment  is 
ordinarily  collected  for  examination  by  means  of  the  centri- 
fuge  (Fig.  97,  above).     An  older  method,  and  one  still   in 

318 


urink:    si  in. m  i.n  is.  319 

vogue  in  some  quarters,  is  the  so-called  gravity  method.  This 
simply  consists  in  placing  the  urine  in  a  conical  glass  and 
allowing  the  sediment  to  settle.  The  collection  of  the  sedi- 
ment by  means  of  the  centrifuge,  however,  is  much  preferable, 
since  the  process  of  sedimentation  may  be  accomplished  by 
the  use  of  this  instrument  in  a  few  minutes,  and  far  more  per- 
fectly, whereas  when  the  other  method  is  used  it  is  frequently 
necessary  to  allow  the  urine  to  remain  in  the  conical  glass 
12-24  hours  before  sufficient  sediment  can  be  secured  for  the 
microscopical  examination. 

(a)   Unorganized  Sediments. 

Ammonium  magnesium  phosphate   ("Triple  phosphate"). 

Calcium  oxalate. 

Calcium  carbonate. 

Calcium  phosphate. 

Calcium  sulphate. 

Uric  acid. 

Urates. 

Cystin. 

Cholesterin. 

Hippuric  acid. 

Leucin  and  tyrosin. 

Hamiatoidin  and  bilirubin. 

Magnesium  phosphate. 

Indigo. 

Xanthin. 

Melanin. 

Ammonium  Magnesium  Phosphate  ("  Triple  Phos- 
phate "). — Crystals  of  "triple  phosphate"  are  a  characteristic 
constituent  of  the  sediment  when  alkaline  fermentation  of  the 
urine  has  taken  place  either  before  or  after  being  voided. 
They  may  even  be  detected  in  amphoteric  or  slightly  acid 
urine  provided  the  ammonium  salts  are  present  in  large 
enough  quantity.  This  substance  may  occur  in  the  sediment 
in  two  forms,  i.  e.,  prisms  and  the  feathery  type.     The  pris- 


320  PHYSIOLOGICAL    CHEMISTRY. 

matic  form  of  crystal  (Fig.  96,  p.  278)  is  the  one  most  com- 
monly observed  in  the  sediment;  the  feathery  form  (Fig. 
96.  p.  278)  predominates  when  the  urine  is  made  ammoniacal 
with  ammonia. 

The  sediment  of  the  urine  in  such  disorders  as  are  accom- 
panied by  a  retention  of  urine  in  the  lower  urinary  tract  con- 
tains "  triple  phosphate "  crystals  as  a  characteristic  constit- 
uent. The  crystals  are  frequently  abundant  in  the  sediment 
during  paraplegia,  chronic  cystitis,  enlarged  prostate  and 
chronic  pyelitis. 

Calcium  Oxalate. — Calcium  oxalate  is  found  in  the  urine 
in  the  form  of  at  least  two  distinct  types  of  crystals,  i.  e.,  the 
dumb-bell  type   and   the   octahedral   type    (Fig.    99,   below). 


Fig.  99. 


Calcium   Oxalate.      (Ogden.) 

Either  form  may  occur  in  the  sediment  of  neutral,  alkaline 
or  acid  urine,  but  both  forms  are  found  most  frequently  in 
urine  having  an  acid  reaction.  Occasionally,  in  alkaline 
urine,  the  octahedral  form  is  confounded  with  "  triple  phos- 
phate "  crystals.  They  may  be  differentiated  from  the  phos- 
phate crystals  by  the  fact  that  they  are  insoluble  in  acetic  acid. 
The  presence  of  calcium  oxalate  in  the  urine  is  not  of  itself 
a  sign  of  any  abnormality,  since  it  is  a  constituent  of  normal 
urine.  It  is  increased  above  the  normal,  however,  in  such 
pathological  conditions  as  diabetes  mellitus,  in  organic  dis- 
eases of  the  liver  and  in  various  other  conditions  which  are 
accompanied  by  a  derangement  of  digestion  or  of  the  oxida- 


URINE  :    SEDIMENTS. 


321 


tion  mechanism,  such  as  occurs  in  certain  diseases  of  the 
heart  and  lungs. 

Calcium  Carbonate. — Calcium  carbonate  crystals  form  a 
typical  constituent  of  the  urine  of  herbivorous  animals.  They 
occur  less  frequently  in  human  urine.  The  reaction  of  urine 
containing  these  crystals  is  nearly  always  alkaline,  although 
they  may  occur  in  amphoteric  or  in  slightly  acid  urine.  It  gen- 
erally crystallizes  in  the  form  of  granules,  spherules  or  dumb- 
bells (Fig.  100,  below).  The  crystals  of  calcium  carbonate  may 
be  differentiated  from  calcium  oxalate  by  the  fact  that  they 
dissolve  in  acetic  acid  with  the  evolution  of  carbon  dioxide 
gas. 

Calcium  Phosphate  (Stellar  Phosphate). — Calcium  phos- 
phate may  occur  in  the  urine  in  three  forms,  i.  e.,  amorphous, 
granular  or  crystalline.     The  crystals  of  calcium  phosphate 

Fig.  100. 


Calcium   Carbonate. 


are  ordinarily  pointed,  wedge-shaped  formations  which  may 
occur  as  individual  crystals  or  grouped  together  in  more  or 
less  regularly  formed  rosettes  (Fig.  j6,  p.  193).  Acid  sodium 
urate  crystals  (Fig.  102,  p.  324)  are  often  mistaken  for  crystals 
of  calcium  phosphate.     We  may  differentiate  between  these 


22 


322  PHYSIOLOGICAL    CHEMISTRY. 

two  crystalline  forms  by  the  fact  that  acetic  acid  will  readily 
dissolve  the  phosphate,  whereas  the  urate  is  much  less  soluble 
and  when  finally  brought  into  solution  and  recrystallized  one 
is  frequently  enabled  to  identify  uric  acid  crystals  which 
have  been  formed  from  the  acid  urate  solution.  The  clinical 
significance  of  the  occurrence  of  calcium  phosphate  crystals 
in  the  urinary  sediment  is  similar  to  that  of  "  triple  phosphate  " 
(seepage  319). 

Calcium  Sulphate. — Crystals  of  calcium  sulphate  are  of 
quite  rare  occurrence  in  the  sediment  of  urine.  Their  pres- 
ence seems  to  be  limited  in  general  to  urines  which  are  of  a 
decided  acid  reaction.  Ordinarily  it  crystallizes  in  the  form 
of  long,  thin,  colorless  needles  or  prisms  (Fig.  95,  page  274) 
which  may  be  mistaken  for  calcium  phosphate  crystals. 
There  need  be  no  confusion  in  this  respect,  however,  since 
the  sulphate  crystals  are  insoluble  in  acetic  acid  which  reagent 
readily  dissolves  the  phosphate.  As  far  as  is  known  their 
occurrence  as  a  constituent  of  urinary  sediment  is  of  very 
little  clinical  significance. 

Uric  Acid. — Uric  acid  forms  a  very  common  constituent 
of  the  sediment  of  urines  which  are  acid  in  reaction.  It 
occurs  in  more  varied  forms  than  any  of  the  other  crystalline 
sediments  (Plate  V,  opposite  page  247,  and  Fig.  101,  page 
323),  some  of  the  more  common  varieties  of  crystals  being- 
rhombic  prisms,  wedges,  dumb-bells,  whetstones,  prismatic 
rosettes,  irregular  rectangular  or  hexagonal  plates,  etc.  Crys- 
tals of  pure  uric  acid  are  always  colorless  (Fig.  89,  page  249), 
but  the  form  occurring  in  urinary  sediments  is  impure  and 
under  the  microscope  appears  pigmented,  the  depth  of  color 
varying  from  light  yellow  to  a  dark  reddish-brown  according 
to  the  size  and  form  of  the  crystal. 

The  presence  of  a  considerable  uric  acid  sediment  does  not, 
of  necessity,  indicate  a  pathological  condition  or  a  urine  of 
increased  uric  acid  content,  since  this  substance  very  often 
occurs  as  a  sediment  in  urines  whose  uric  acid  content  is 
diminished  from  the  normal  merely  as  a  result  of  changes  in 


(  ui  \ ■  i-  :    si'  hi  m  i  NTS. 


[23 


reaction,  etc.  Pathologically,  uric  acid  sediments  occur  in 
gout,  acute  febrile  conditions,  chronic  interstitial  nephritis, 
etc.  If  the  microscopical  examination  is  not  conclusive,  uric 
acid  may  be  differentiated  from  Other  crystalline  urinary  sedi- 


FlG.    IOI. 


Various  Forms  of  Uric  Acid. 
i.   Rhombic   plates;    2,   whetstone   forms;    3,    3,   quadrate   forms;    4,    5,   pro- 
longed  into   points ;   6,   8.   rosettes ;    7.   pointed   bundles ;   9,   barrel   forms   pre- 
cipitated by  adding  hydrochloric   acid  to   urine. 


ments  from  the  fact  that  it  is  soluble  in  alkalis,  alkali  carbo- 
nates, boiling  glycerin,  concentrated  sulphuric  acid  and  in  cer- 
tain organic  bases  such  as  ethylamine  and  piperidin.  It  also 
responds  to  the  murexid  test  (see  page  249)  and  to  Schiff's 
reactiqn  (see  page  249). 

Urates. — The  urate  sediment  may  consist  of  a  mixture 
of  the  urates  of  ammonium,  calcium,  magnesium,  potassium 
and  sodium.  The  ammonium  urate  may  occur  in  neutral, 
alkaline  or  acid  urine,  whereas  the  other  forms  of  urates  are 
confined  to  the  sediments  of  acid  urines.  Sodium  urate 
occurs  in  sediments  more  abundantly  than  the  other  urates. 


324 


PHYSIOLOGICAL    CHEMISTRY, 


The  urates  of  calcium,  magnesium  and  potassium  are  amor- 
phous in  character,  whereas  the  urate  of  ammonium  is  crystal- 
line. Sodium  urate  may  be  either  amorphous  or  crystalline. 
When  crystalline  it  forms  groups  of  fan-shaped  clusters  or 
colorless,  prismatic  needles  (Fig.  102,  below).  Ammonium 
urate  is  ordinarily  present  in  the  sediment  in  the  burr-like 
form  of  the  "thorn-apple"  crystal,  i.  e.,  yellow  or  reddish- 
brown  spheres,  covered  with  sharp  spicules  or  prisms  (Plate 
VI,  opposite  page  324).     The  urates  are  all  soluble  in  hydro- 

FlG.    102. 


Acid  Sodium  Urate. 


chloric  acid  or  acetic  acid  and  their  acid  solutions  yield  crystals 
of  uric  acid  upon  standing.  They  also  respond  to  the  murexid 
test.  The  clinical  significance  of  urate  sediments  is  very  simi- 
lar to  that  of  uric  acid.  A  considerable  sediment  of  amor- 
phous urates  does  not  necessarily  indicate  a  high  uric  acid 
content,  but  ordinarily  signifies  a  concentrated  urine  having 
a  very  strong  acidity. 

Cystin. — Cystin  is  one  of  the  rarer  of  the  crystalline  uri- 
nary sediments.  It  has  been  claimed  that  it  occurs  more 
often  in  the  urine  of  men  than  of  women.  Cystin  crystal- 
lizes in  the  form  of  thin,  colorless,  hexagonal  plates  (Fig.  32, 


PLATE    VI. 


Ammonium    Urate,  showing  Spherules  and  Thorn  vpple-shaped  Crystals 
(From  Ogden,  alter  Peyer.) 


urine:  sediments.  325 

p.  76,  and  Fig.  [03,  bel<»\v)  which  arc  insoluble  in  water,  alco- 
hol and  acetic  acid  and  soluble  in  minerals  acids,  alkalis  and 
especially  in  ammonia.  Cystin  may  be  identified  by  burn- 
ing it  upon  platinum  foil  under  which  condition  it  does  not 
melt  but  vields  a  bluish-green  flame. 


o 


I 


Fig.  103. 

• 


Cvstix.     (Ogden.) 

Cholesterin. — Cholesterin  crystals  have  been  but  rarely 
detected  in  urinary  sediments.  When  present  they  probably 
arise  from  a  pathological  condition  of  some  portion  of  the 
urinary  tract.  Crystals  of  cholesterin  have  been  found  in 
the  sediment  in  cystitis,  pyelitis,  chyluria  and  nephritis.  Or- 
dinarily it  crystallizes  in  large  regular  and  irregular  colorless, 
transparent  plates,  some  of  which  possess  notched  corners 
(Fig.  42,  page  125).  Frequently,  instead  of  occurring  in  the 
sediment,  it  is  found  in  the  form  of  a  film  on  the  surface  of 
the  urine. 

Hippuric  Acid. — This  is  one  of  the  rarer  sediments  of 
human  urine.  It  deposits  under  conditions  similar  to  those 
which  govern  the  formation  of  uric  acid  sediments.  The 
crystals,  which  are  colorless  needles  or  prisms  (Fig.  92.  page 
256)  when  pure,  are  invariably  pigmented  in  a  manner  similar 
to  the  uric  acid  crystals  when  observed  in  urinary  sediment 
and  because  of  this  fact  are  frequently  confounded  with  the 
rarer  forms  of  uric  acid.  Hippuric  acid  may  be  differen- 
tiated from  uric  acid  from  the  fact  that  it  does  not  respond 
to  the  murexid  test  and  is  much  more  soluble  in  water  and 
in  ether.     The  detection  of  crystals  of  hippuric  acid  in  the 


326  PHYSIOLOGICAL    CHEMISTRY. 

urine  has  very  little  clinical  significance,  since  its  presence  in 
the  sediment  depends  in  most  instances  very  greatly  upon  the 
nature  of  the  diet.  It  is  particularly  prone  to  occur  in  the 
sediment  after  the  ingestion  of  certain  fruits  as  well  as  after 
the  ingestion  of  benzoic  acid  (see  page  256). 

Leucin  and  Tyrosin. — Leucin  and  tyrosin  have  frequently 
been  detected  in  the  urine,  either  in  solution  or  as  a  sediment. 
Neither  of  them  occurs  in  the  urine  ordinarily  except  in  asso- 
ciation with  the  other,  i.  e.,  whenever  leucin  is  detected  it 
is  more  than  probable  that  tyrosin  accompanies  it.  They 
have  been  found  pathologically  in  the  urine  in  acute  yellow 
atrophy  of  the  liver,  in  acute  phosphorus  poisoning,  in  cir- 
rhosis of  the  liver,  in  severe  cases  of  typhoid  fever  and  small- 
pox, and  in  leukaemia.  In  urinary  sediments  leucin  ordi- 
narily  crystallizes    in    characteristic    spherical    masses    which 

show  both  radial  and  concen- 

FlG.    I04.  .  ...  ,  1-11 

trie  striations  and  are  highly 
refractive  (Fig.  104,  p.  326). 
For  the  crystalline  form  of  pure 
leucin  obtained  as  a  decompo- 
sition product  of  proteicl  see 
Fig.  24,  p.  69.  Tyrosin  crys- 
tallizes in  urinary  sediments  in 
the  well  known  sheaf  or  tuft 
W  formation    (Fig.    23,    p.    68). 

Crystals    of    Impure    Leucin.         por    otlier    tests    on    leucill    and 

(Ogden.) 

tyrosin  see  pages  80  to  82. 
Haematoidin  and  Bilirubin. — There  are  divergent  opin- 
ions regarding  the  occurrence  of  these  bodies  in  urinary  sedi- 
ment. Each  of  them  crystallizes  in  the  form  of  tufts  of 
small  needles  or  in  the  form  of  small  plates  which  are  ordi- 
narily yellowish-red  in  color  (Fig.  41,  p.  119).  Because  of 
the  fact  that  the  crystalline  form  of  the  two  substances  is 
identical  many  investigators  claim  them  to  be  one  and  the 
same  body.  Other  investigators  claim,  that  while  the  crystal- 
line form  is  the  same  in  each  case,  that  there  are  certain  chem- 


i  rink:    SEDIMENTS.  327 

ical  differences  which  may  he  brought  out  very  strikingly  Im- 
properly testing.  For  instance,  it  has  heen  claimed  that 
hsematoidin  may  he  differentiated  from  bilirubin  through  the 
fact  that  it  gives  a  momentary  color  reaction  (hlue)  when 
nitric  acid  is  brought  in  contact  with  it.  and  further,  that 
it  is  not  dissolved  on  treatment  with  ether  or  potassium 
hydroxide.  Pathologically,  typical  crystals  of  hsematoidin 
or  bilirubin  have  been  found  in  the  urinary  sediment  in  jaun- 
dice, acute  yellow  atrophy  of  the  liver,  carcinoma  of  the  liver, 
cirrhosis  of  the  liver,  and  in  phosphorus  poisoning,  typhoid 
fever  and  scarlatina. 

Magnesium  Phosphate. — Magnesium  phosphate  crystals 
occur  rather  infrequently  in  the  sediment  of  urine  which  is 
neutral,  alkaline  or  feebly  acid  in  reaction.  It  ordinarily 
crystallizes  in  elongated,  highly  refractive,  rhombic  plates 
which  are  soluble  in  acetic  acid. 

Indigo. — Indigo  crystals  are  frequently  found  in  urine 
which  has  undergone  alkaline  fermentation.  They  result 
from  the  breaking  down  of  indoxyl-sulphates  or  indoxyl- 
glycuronates.  Ordinarily  indigo  deposits  as  dark  blue  stel- 
late needles  or  occurs  as  amorphous  particles  or  broken  frag- 
ments. These  crystalline  or  amorphous  forms  may  occur  in  the 
sediment  or  may  form  a  blue  film  on  the  surface  of  the  urine. 
Indigo  crystals  generally  occur  in  urine  which  is  alkaline  in 
reaction,  but  they  have  been  detected  in  acid  urine. 

Xanthin. — Xanthin  is  a  constituent  of  normal  urine  but 
is  found  in  the  sediment  in  crystalline  form  very  infrequently, 
and  then  only  in  pathological  urine.  When  present  in  the 
sediment  xanthin  generally  occurs  in  the  form  of  whetstone- 
shaped  crystals  somewhat  similar  in  form  to  the  whetstone  va- 
riety of  uric  acid  crystal.  They  may  be  differentiated  from  uric 
acid  by  the  great  ease  with  which  they  may  be  brought  into 
solution  in  dilute  ammonia  and  on  applying  heat.  Xanthin 
may  also  form  urinary  calculi.  The  clinical  significance  of 
xanthin  in  urinary  sediment  is  not  well  understood. 

Melanin. — Melanin    is    an    extremelv    rare   constituent   of 


328  PHYSIOLOGICAL    CHEMISTRY. 

urinary  sediments.  Ordinarily  in  melanuria  the  melanin  re- 
mains in  solution;  if  it  separates  it  is  generally  held  in  sus- 
pension as  fine  amorphous  granules. 

(b)   Organized  Sediments. 

Epithelial  cells. 
Pus  cells. 

'  Hyaline. 

Granular. 

Epithelial. 
Casts  1  Blood. 

Fatty. 

Waxy. 

Pus. 
Cylindroids. 
Erythrocytes. 
Spermatozoa. 
Urethral  filaments. 
Tissue  debris. 
Animal  parasites. 
Micro-organisms. 
Fibrin. 
Foreign  substances  clue  to  contamination.. 

Epithelial  Cells. — The  detection  of  a  certain  number 
of  these  cells  in  urinary  sediment  is  not,  of  itself,  a  patho- 
logical sign,  since  they  occur  in  normal  urine.  However,  in 
certain  pathological  conditions  they  are  greatly  increased  in 
number,  and  since  different  areas  of  the  urinary  tract  are 
lined  with  different  forms  of  epithelial  cells,  it  becomes  nec- 
essary, when  examining  urinary  sediments,  to  note  not  only 
the  relative  number  of  such  cells,  but  at  the  same  time  to 
carefully  observe  the  shape  of  the  various  individuals  in 
order  to  determine,  as  far  as  possible,  from  what  portion  of 
the  tract  they  have  been  derived.  Since  the  different  layers 
of  the  epithelial  lining  are  composed  of  cells  different  in  form 
from  those  of  the  associated  layers,  it  is  evident  that  a  careful 
microscopical  examination  of  these  cells  may  tell  us  the  par- 


urine:  sediments. 


329 


ticular  layer  which  is  being  desquamated.  It  is  frequently  a 
most  difficult  undertaking',  however,  to  make  a  clear  differen- 
tiation between  the  various  forms  of  epithelial  cells  present 
in  a  sediment.  If  skilfully  done,  such  a  microscopical  differ- 
entiation may  prove  to  be  of  very  great  diagnostic  aid. 

The  principal  forms  of  epithelial  cells  met  with  in  urinary 
sediments  are  shown  in  Fig.  105,  below. 

Fig.  105. 


Epithelium  from  Different  Areas  of  the  Urinary  Tract. 
a,  Leucocyte  (for  comparison);  b,  renal  cells;  c,  superficial  pelvic  cells;  d, 
deep  pelvic  cells ;  e,  cells  from  calices  ;  /,  cells  from  ureter ;  g,  g,  g,  g,  g,  squa- 
mous epithelium  from  the  bladder  ;  /;,  h,  neck-of-bladder  cells ;  i,  epithelium 
from  prostatic  urethra  ;  k,  urethral  cells ;  /,  /,  scaly  epithelium ;  m,  in',  cells 
from  seminal  passages ;  n,  compound  granule  cells ;  o,  fatty  renal  cell. 
(Ogden.) 

Pus  Cells. — Pus  corpuscles  or  leucocytes  are  present  in  ex- 
tremely small  numbers  in  normal  urine.  Any  considerable 
increase  in  the  number,  however,  ordinarily  denotes  a  patho- 
logical condition,  generally  an  acute  or  chronic  inflammatory 
condition  of  some  portion  of  the  urinary  tract.  The  sudden 
appearance  of  a  large  amount  of  pus  in  a  sediment  denotes 
the  opening  of  an  abscess  into  the  urinary  tract.  Other  form 
elements,  such  as  epithelial  cells,  casts,  etc.,  ordinarily  accom- 


33°  PHYSIOLOGICAL    CHEMISTRY. 

pany  pus  corpuscles  in  urinary  sediment  and  a  careful  exami- 
nation of  these  associated  elements  is  necessary  in  order  to 
form  a  correct  diagnosis  as  to  the  origin  of  the  pus.  Proteid 
is  always  present  in  urine  which  contains  pus. 

Fig.  106. 


Pus   Corpuscles.     (After   Ultsmann.) 
i.    Normal;    2,    showing    amoeboid    movements;    3,    nuclei    rendered    distinct 
by  acetic  acid;   4,  as  observed  in   chronic  pyelitis;    5,   swollen  by  ammonium 
carbonate. 

The  appearance  which  pus  corpuscles  exhibit  under  the 
microscope  depends  greatly  upon  the  reaction  of  the  urine 
containing  them.  In  acid  urine  they  generally  present  the 
appearance  of  round,  colorless  cells  composed  of  refractive, 
granular  protoplasm,  and  may  frequently  exhibit  amoeboid 
movements,  especially  if  the  slide  containing  them  be  warmed 
slightly.  They  are  nucleated  (one  or  more  nuclei),  the  nuclei 
being  clearly  visible  only  upon  treating  the  cells  with  water, 
acetic  acid  or  some  other  suitable  reagent.  In  urine  which  has 
a  decided  alkaline  reaction,  on  the  other  hand,  the  pus  corpus- 
cles are  often  greatly  degenerated.  They  may  be  seen  as 
swollen,  transparent  cells,  which  exhibit  no  granular  structure 
and  as  the  process  of  degeneration  continues  the  cell  outline 
ceases  to  be  visible,  the  nuclei  fade,  and  finally  only  a  mass 


urine:  sediments.  331 

of  debris  containing  isolated   nuclei   and   an  occasional  cell 
remains. 

It  is  frequently  rather  difficult  to  make  a  differentiation 
between  pus  corpuscles  and  certain  types  of  epithelial  cells 
which  arc  similar  in  form.  Such  confusion  may  be  avoided 
by  the  addition  <>t"  iodine  solution  (I  in  KI  ),  a  reagent  which 
stains  the  pus  corpuscles  a  deep  mahogany-brown  and  trans- 
mits to  the  epithelial  cells  a  light  yellow  tint.  The  test  pro- 
posed by  Vitali  often  gives  very  satisfactory  results.  This 
simply  consists  in  acidifying-  the  urine  (if  alkaline)  with 
acetic  acid,  then  filtering,  and  treating  the  sediment  on  the 
filter  paper  with  freshly  prepared  tincture  of  guaiac.  The 
presence  of  pus  in  the  sediment  is  indicated  if  a  blue  color  is 
observed.  Large  numbers  of  pus  corpuscles  are  present  in 
the  urinary  sediment  in  gonorrhoea,  leucorrhcea,  chronic 
pyelitis  and  in  abscess  of  the  kidney. 


Fig.  107. 


Hyaline   Casts. 
One  cast  is  impregnated  with  four  renal  cells. 


33* 


PHYSIOLOGICAL    CHEMISTRY, 


Casts. — These  are  cylindrical  formations,  which  originate 
in  the  uriniferous  tubules  and  are  forced  out  by  the  pressure 
of  the  urine.  They  vary  greatly  in  size  but  in  nearly  every 
instance  they  possess  parallel  sides  and  rounded  ends.  The 
finding  of  casts  in  the  urine  is  very  important  because  of  the 
fact  that  they  generally  indicate  some  kidney  disorder ;  if  albu- 
min accompanies  the  casts  the  indication  is  much  accentuated. 
Casts  have  been  classified  according  to  their  microscopical  char- 
acteristics as  follows:  (a)  Hyaline,  (b)  granular,  (c)  epi- 
thelial, (d)   blood,  (e)    fatty,  (/)   waxy,  (g)   pus. 

(a)  Hyaline  Casts. — These  are  composed  of  a  basic  material 
which  is  transparent,  homogeneous  and  very  light  in  color 
(Fig.  107,  p.  331).     In  fact,  chiefly  because  of  these  physical 

Fig.  108. 


Granular   Casts.      (After  Peyer.) 


properties,  they  are  the  most  difficult  form  of  renal  cast  to 
detect  under  the  microscope.  Frequently  such  casts  are  im- 
pregnated with  deposits  of  various  forms  such  as  erythrocytes, 
epithelial  cells,  fat  globules,  etc.,  thus  rendering  the  form  of 


URINE  :    SEDIMENTS. 


333 


the  cast  more  plainly  visible.     Staining  is  often  resorted  to  in 

order  to  render  the  shape  and  character  of  the  casl  more  easily 
determined.  Ordinary  iodine  solution  (1  in  K  I  )  may  be  used 
in  this  connection;  many  of  the  anilin  dyes  arc  also  in  common 
use  for  this  purpose,  e.  g.}  gentian-violet,  Bismarck-brown, 
methylene-blue.  fuchsin  and  eosin.  Generally,  hut  not  always, 
albumin  is  present  in  urine  containing  hyaline  casts.  Hyaline 
casts  are  common  t<>  all  kidney  disorders,  but  occur  particu- 
larly in  the  earliest  and  recovering  stages  of  parenchymatous 
nephritis  and  in  interstitial  nephritis. 

(b)  Granular  Casts. — The  common  hyaline  material  is  ordi- 
narily the  basic  substance  of  this  form  of  cast.  The  granular 
material  generally  consists  of  albumin,  epithelial  cells,  fat  or 


Fig.  109. 


Fig.  1 10. 


Granular   Casts. 

a,    Finely     granular ;     b,    coarsely 

granular. 


Epithelial   Casts. 


disintegrated  erythrocytes  or  leucocytes,  the  character  of  the 
cast  varying  according  to  the  nature  and  size  of  the  granules 
(Fig.  108,  page  332,  and  Fig.  109,  above).  Thus  we  have 
casts  of  this  general  type  classified  as  finely  granular  and 
coarsely  granular  casts.  Granular  casts,  and  in  particular  the 
finely  granular  types,  occur  in  the  sediment  in  practically  every 
kidney  disorder  but  are  probably  especially  characteristic  of 
the  sediment  in  inflammatory-  disorders. 


334 


PHYSIOLOGICAL    CHEMISTRY.- 


(c)  Epithelial  Casts. — These  are  casts  bearing  upon  their 
surface  epithelial  cells  from  the  lining  of  the  uriniferous 
tubules  (Fig.  no,  p.  333).  The  basic  material  of  this  form  of 
cast  may  be  hyaline  or  granular  in  nature.  Epithelial  casts 
are  particularly  abundant  in  the  urinary  sediment  in  acute 
nephritis. 

(d)  Blood  Casts. — Casts  of  this  type  may  consist  of  ery- 
throcytes borne  upon  a  hyaline  or  a  fibrinous  basis  (Fig.  Ill, 
below).     The  occurrence  of  such  casts  in  the  urinary  sediment 

Fig.  hi. 


Blood,    Pus,   Hyaline  and    Epithelial    Casts. 
a,  Blood  casts  ;   b,  pus  cast ;   c,  hyaline  cast  impregnated  with  renal  cells ;   d, 

epithelial  casts. 

denotes  renal  haemorrhage  and  they  are  considered  to  be 
especially  characteristic  of  acute  diffuse  nephritis  and  acute 
congestion  of  the  kidney. 

(e)  Fatty  Casts. — Fatty  casts  may  be  formed  by  the  deposi- 
tion of  fat  globules  or  crystals  of  fatty  acid  upon  the  surface 
of  a  hyaline  or  granular  cast  (Fig.  112,  p.  335).     In  order  to 


TRIM:  :    SEDIMENTS. 


335 


Fatty  Casts.     (After  Peyer.) 
Fig.  113. 


a,  Fatty  casts ;  b,  waxy  casts. 
Fatty  and  Waxy  Casts. 


336  PHYSIOLOGICAL    CHEMISTRY. 

constitute  a  true  fatty  cast  the  deposited  material  must  cover 
the  greater  part  of  the  surface  area  of  the  cast.  The  presence 
of  fatty  casts  in  urinary  sediment  indicates  fatty  degeneration 
of  the  kidney ;  such  casts  are  particularly  characteristic  of  sub- 
acute and  chronic  inflammations  of  the  kidney. 

Fig.  114. 


Cylindroids.      (After  Peyer.) 

(f)  Waxy  Casts. — These  casts  possess  a  basic  substance 
similar  to  that  which  enters  into  the  foundation  of  the  hyaline 
form  of  cast.  In  common  with  the  hyaline  type  they  are  color- 
less, refractive  bodies  but  differ  from  this  form  of  cast  in  be- 
ing, in  general,  of  greater  length  and  diameter  and  possessing 
sharper  outlines  and  a  light  yellow  color  (Fig.  113,  p.  335). 
Such  casts  occur  in  several  forms  of  nephritis  but  do  not 
appear  to  characterize  any  particular  type  of  the  disorder  ex- 
cept amyloid  disease,  in  which  they  are  rather  common. 

(g)  Pus  Casts. — Casts  whose  surface  is  covered  with  pus 
cells  or  leucocytes  are  termed  pus  casts  (Fig.  m,p.  334.  They 
are  frequently  mistaken  for  epithelial  casts.     The  differentia- 


URINE  :    SEDIMENTS. 


337 


tion  between  these  two  types  is  made  very  simple  however 
by  treating-  the  cast  with  acetic  acid  which  causes  the  nuclei  of 
the  leucocytes  to  become  plainly  visible.  The  true  pus  cast  is 
quite  rare  and  indicates  renal  suppuration. 

Cylindroids. — These  formations  may  occur  in  normal  or 
pathological  urine  and  have  no  particular  clinical  significance. 
They  are  frequently  mistaken  for  true  casts,  especially  the  hya- 
line type,  but  they  are  ordinarily  flat  in  structure  with  a  rather 
smaller  diameter  than  casts,  may  possess  forked  or  branching 
ends  and  are  not  composed  of  homogeneous  material  as  are  the 
hyaline  casts.  Such  "  false  casts  "  may  become  coated  with 
urates,  in  which  event  they  appear  granular  in  structure.  The 
basic  substance  of  cylindroids  is  often  the  nucleo-proteid  of 
the  urine  (see  Fig.   114.  page  336). 

Erythrocytes. — These  form  elements  are  present  in  the 
urinary  sediment  in  various  diseases.     They  may  appear  as 

Fig.  11  v 


Crenated  Erythrocytes. 


the  normal  biconcave,  yellow  erythrocyte  (Plate  IV,  opposite 
page  151)  or  may  exhibit  certain  modifications  in  form  such  as 
the  crenated  type  (Fig.  115,  above)  which  is  often  seen  in  con- 
23 


338 


PHYSIOLOGICAL    CHEMISTRY. 


centrated  urine.  Under  different  conditions  they  may  become 
swollen  sufficiently  to  entirely  erase  the  biconcave  appearance 
and  may  even  occur  in  the  form  of  colorless  spheres  having  a 
smaller  diameter  than  the  origina  1  disc-shaped  corpuscles. 
Erythrocytes  are  found  in  urinary  sediment  in  hemorrhage  of 
the  kidney  or  of  the  urinary  tract,  in  traumatic  hemorrhage, 
hemorrhage  from  congestion  and  in  hemorrhagic  diathesis. 

Spermatozoa. — Spermatozoa  may  be  detected  in  the  urinary 
sediment  in  diseases  of  the  genital  organs,  as  well  as  after 
coitus,    nocturnal    emissions,    epileptic    and    other    convulsive 

Fig.  i  i  6. 


Human    Spermatozoa. 

attacks  and  sometimes  in  severe  febrile  disorders,  especially  in 
typhoid  fever.  In  form  they  consist  of  an  oval  body,  to  which 
is  attached  a  long,  delicate  tail  (Fig.  116,  above).  Upon  ex- 
amination they  may  show  motility  or  may  be  motionless. 

Urethral  Filaments. — These  are  peculiar  thread-like  bodies 
which  are  sometimes  found  in  urinary  sediment.  They  may 
occasionally  be  detected  in  normal  urine  and  pathologically  are 
found  in  the  sediment  in  acute  and  chronic  gonorrhoea  and  in 
urethrorrhcea.  The  ground-substance  of  these  urethral  fila- 
ments is  in  part,  at  least,  similar  to  that  of  the  cylindroids  (see 


urine:  sediments.  339 

page  337)*  The  urine  first  voided  in  the  morning  is  besl 
adapted   for  the  examination   for  filaments.     These  filaments 

may  ordinarily  l>e  removed  l>y  a  pipette  since  they  are  gener- 
ally macr<  >scopic. 

Tissue  Debris. — Masses  of  cells  or  fragments  of  tissue  arc 
frequently  found  ill  urinary  sediment.  They  may  be  found  in 
the  sediment  in  tubercular  affections  of  the  kidney  and  urinary 
tract  or  in  tumors  of  these  organs.  Ordinarily  it  is  necessary 
to  make  a  histological  examination  of  such  tissue  fragments 
before  coming  t<»  a  final  decision  as  to  their  origin. 

Animal  Parasites. — The  cysts,  booklets  and  membrane 
shreds  of  echinococci  are  sometimes  found  in  urinary  sedi- 
ments. Other  animal  organisms  which  are  more  rarely  met 
with  in  the  urine  are  embryos  of  the  Filaria  sanguinis  and 
eggs  of  the  Distoma  h<cmatobium  and  Ascarides.  Animal 
parasites  in  general  occur  most  frequently  in  the  urine  in  trop- 
ical countries. 

Micro-Organisms. — Bacteria  as  well  as  yeast  and  moulds 
are  frequently  detected  in  the  urine.  Both  the  pathogenic  and 
non-pathogenic  forms  of  bacteria  may  occur.  The  non-patho- 
genic forms  most  frequently  observed  are  micrococcus  urea, 
bacillus  urea",  and  staphylococcus  urccc  liquefacicus.  Of  the 
pathogenic  forms  many  have  been  observed,  c.  g.,  Bacterium 
Coli,  typhoid  bacillus,  tubercle  bacillus,  gonococcus,  bacillus 
pyocyaucus  and  proteus  vulgaris.  Yeast  and  moulds  are  most 
frequently  met  with  in  diabetic  urine. 

Fibrin. — Following  hematuria,  fibrin  clots  are  occasionally 
observed  in  the  urinary  sediment.  They  are  generally  of  a 
semi-gelatinous  consistency  and  of  a  very  light  color,  and  when 
examined  under  the  microscope  they  are  seen  to  be  composed 
of  bundles  of  highly  refractive  fibres  which  run  parallel. 

Foreign  Substances  Due  to  Contamination. — Such  for- 
eign substances  as  fibers  of  silk,  linen  or  wool ;  starch  granules, 
hair,  fat  and  sputum,  as  well  as  muscle  fibers,  vegetable  cells 
and  food  particles  are  often  found  in  the  urine.  Care  should 
be  taken  that  these  foreign  substances  are  not  mistaken  for  any 
of  the  true  sedimentary  constituents  already  mentioned. 


CHAPTER    XX. 
URINE:    CALCULI. 

Urinary  calculi,  also  called  concretions,  or  concrements  are 
solid  masses  of  urinary  sediment  formed  in  some  part  of  the 
urinary  tract.  They  vary  in  shape  and  size  according  to  their 
location,  the  smaller  calculi  termed  sand  or  gravel  in  general 
arising  from  the  kidney  or  the  pelvic  portion  of  the  kidney, 
whereas  the  large  calculi  are  ordinarily  formed  in  the  bladder. 
There  are  two  general  classes  of  calculi  as  regards  composition, 
i.  e.,  simple  and  compound.  The  simple  form  is  made  up  of 
but  a  single  constituent  whereas  the  compound  type  contains 
two  or  more  individual  constituents.  The  structural  plan  of 
most  calculi  consists  of  an  arrangement  of  concentric  rings 
about  a  central  nucleus,  the  number  of  rings  frequently  being 
dependent  upon  the  number  of  individual  constituents  which 
enter  into  the  structure  of  the  calculus.  In  case  two  or  more 
calculi  unite  to  form  a  single  calculus  the  resultant  body  will 
obviously  contain  as  many  nuclei  as  there  were  individual 
calculi  concerned  in  its  construction.  Under  certain  conditions 
the  growth  of  a  calculus  will  be  principally  in  onlv  one  direc- 
tion thus  preventing  the  nucleus  from  maintaining  a  central 
location.  The  qualitative  composition  of  urinary  calculi  is 
dependent,  in  great  part,  upon  the  reaction  of  the  urine  c.  g., 
if  the  reaction  of  the  urine  is  acid  the  calculi  present  will  be 
composed,  in  great  part  at  least,  of  substances  that  are  capable 
of  depositing  in  acid  urine. 

According  to  Ultzmann,  out  of  545  cases  of  urinary  calcu- 
lus, uric  acid  and  urates  formed  the  nucleus  in  about  81  per 
cent  of  the  cases ;  earthy  phosphates  in  about  9  per  cent ;  calcium 
oxalate  in  about  6  per  cent ;  cystin  in  something  over  1  per  cent, 
while  in  about  3  per  cent  of  the  cases  some  foreign  body  com- 
prised the  nucleus. 

340 


URINI".  :    CALCULI.  341 

In  the  chemical  examination  of  urinary  calculi  the  1 
valuable  data  are  obtained  by  subjecting  each  of  the  concentric 
layers  of  the  calculus  to  a  separate  analysis.  Material  for  ex- 
amination may  be  conveniently  obtained  by  sawing  the  calculus 
carefully  through  the  nucleus,  then  separating  the  various  lay- 
ers or  by  scraping  off  from  each  layer  1  without  separating  the 
lavers)  enough  powder  to  conduct  the  examination  as  outlined 
in  the  scheme  (see  page  343). 

Varieties  of  Caculus. 

Uric  Acid  and  Urate  Calculi. — Uric  acid  and  urates  consti- 
tute the  nuclei  of  a  large  proportion  (81  per  cent)  of  urinary 
concretions.  Such  stones  are  always  colored,  the  tint  varying 
from  a  pale  yellow  to  a  brownish-red.  The  surface  of  such 
calculi  is  generally  smooth  but  it  may  be  rough  and  uneven. 

Phosphatic  Calculi. — Ordinarily  these  concretions  consist 
principally  of  "triple  phosphate"  and  other  phosphates  of  the 
alkaline  earths,  with  very  frequent  admixtures  of  urates  and 
oxalates.  The  surface  of  such  calculi  is  generally  rough  but 
may  occasionally  be  rather  smooth.  The  calculi  are  somewhat 
variable  in  color  exhibiting  gray,  white  or  yellow  tints  under 
different  conditions.  When  composed  of  earthy  phosphates 
the  calculi  are  characterized  by  their  friability. 

Calcium  Oxalate  Calculi. — This  is  the  hardest  form  of 
calculus  to  deal  with,  and  is  rather  difficult  to  crush.  They 
ordinarily  occur  in  two  general  forms,  i.  e.,  the  small,  smooth 
concretion  which  is  characterized  as  the  hemp-seed  calculus 
and  the  medium-sized  or  large  stone  possessing  an  extremely 
uneven  surface  which  is  generally  classed  as  a  mulberry  calcu- 
lus. This  roughened  surface  of  the  latter  form  of  calculus  is 
due,  in  many  instances,  to  protruding  calcium  oxalate  crystals 
of  the  octahedral  type. 

Calcium  Carbonate  Calculi. — Calcium  carbonate  concre- 
tions are  quite  common  in  herbivorous  animals  but  of  exceed 
ingly  rare  occurrence  in  man.     They  are  generally  small,  white 
or  grayish  calculi,  spherical  in  form  and  possess  a  hard,  smooth 
surface. 


342  PHYSIOLOGICAL    CHEMISTRY. 

Cystin  Calculi. — The  cystin  calculus  is  a  rare  variety  of 
calculus.  Ordinarily  they  occur  as  small,  smooth,  oval  or 
cylindrical  concretions  which  are  white  or  yellow  in  color 
and  of  a  rather  soft  consistency. 

Xanthin  Calculi. — This  form  of  calculus  is  somewhat  more 
rare  than  the  cystin  type.  The  color  may  vary  from  white  to 
brownish-yellow.  Very  often  uric  acid  and  urates  are  asso- 
ciated with  xanthin  in  this  type  of  calculus.  Upon  rubbing  a 
xanthin  calculus  it  has  the  property  of  assuming  a  wax-like 
appearance. 

Urostealith  Calculi. — This  form  of  calculus  is  extremely 
rare.  Such  concretions  are  composed  principally  of  fat  and 
fatty  acid.  When  moist  they  are  soft  and  elastic  but  when 
dried  they  become  brittle.  Urostealiths  are  generally  light  in 
color. 

Fibrin  Calculi. — Fibrin  calculi  are  produced  in  the  process 
of  blood  coagulation  within  the  urinary  tract.  They  fre- 
quently occur  as  nuclei  of  other  forms  of  calculus.  They  are 
rarely  found. 

Cholesterin  Calculi. — An  extremely  rare  form  of  calculus 
somewhat  resembling  the  cystin  type. 

Indigo  Calculi. — Indigo  calculi  are  extremely  rare,  only 
two  cases  having  been  reported.  One  of  these  indigo  calculi 
is  on  exhibition  in  the  museum  of  Jefferson  Medical  College 
of  Philadelphia. 

The  scheme,  proposed  by  Heller  and  given  on  page  343,  will 
be  found  of  much  assistance  in  the  chemical  examination  of 
urinary  calculi. 


urine:  calculi. 


343 


i  in  1  [eating 

the  Powdei  on  Platinum  Foil,  It 

Does  not  burn 

Does  hum 

The   powder  when   treated  with  11*1 

With  flange 

Without  flame 

a  not  effervesce 

3"  ~ 

2 
(I 

J  a 

-i 

The  pow 
der  ^ivi  -  th< 
murexid  test 

The  gently-heated  powder  with  HC1 

3-2 

™  3 

3 

a  3 

§•0^ 

S  5 

p  r» 

ft 

—  n 

STa 

Tin    powder  when   moistened 

•  >< 

ft 

V3 

-■  g 

The  pow- 

With a  little  K<  »ll 

?o 

5" 

!?  rt 

E   i- 

der  when 

o  - 

s< 

'    — 

n    < 

en    ft 

treate<l  with 

p 

a  § 

— 

Da 

•     0 
- 

U3 

p  = 

2  5' 

a.  c 

—  5 

— 
p_ 

ft* 

o 
o 

^  IT 

P 

2  c 
rt  3 

o  «■• 

S  3 
P   c 
3    3 
ft   £ 
1^ 

KOI1  gives 

-.  — 

T3    P 

-  § 

2.  3 

-     z 

.,     3 

Z     ■_' 

~  9 

ft   " 

IE 

5' 
c 
o 
c 
u 

=    B 

3"     / 

n   3- 
-3    S 

5:  ft 
p  r 

-     — 

3.'  3 

g  S" 

5'" 

3     (B 

p  ° 
p  — > 

n    

O 
a. 

o 

3  — 
g  5 

3    rt 

-■  3* 
ft 

r— 

p  o 

S.  _: 

—  !T 

i     B 
3    <n 

ft     r» 

n  - 

as 

°    3 
P    " 

o 

0 
— > 

0 

c 

a.  a 

p>  ft 

E.  M 

5  5 

3    ft 

f  s 

ft     — 

2-S? 

eg 

2  S1 

3  « 

►1 

ft 
IB 

S" 

0 

51 

p 

cn 

o 

3 

—  n 
En 

|    0 

- 
< 

ft 

w 

ft 

< 
ft 

orq   *" 

ft 

3    _ 

ft    £"* 
-l     3 

P       y! 

<n 

— 
p" 

S*  3" 

§  2 
o"3 

5  3' 

3     _ 
3.  _ 

3 

3 

crq 
p 

O 

ft 

p 

g; 

< 
■ 

5' 

o 

3     B 

q  3 

3.5 

11  5 

--^   3 

IF 

r. 
n 

/ 

n 
ft 

-   0 

2"  c 
3  cr 

2,  <T 

=  =' 
«    p 

o 

o 
p 

V 

3 
B, 

p  ■ 

i  = 

°  sr 

3    ft 

era  § 

CO 

B- 
o 

3 
3 
o 

3 

p' 

"-t 
ft 

ft 
p 
5 
3 
o 

3 

c 

'-/"-  r* 

3' 

c 

P 

p' 

P 

o 

a 

t/3      '— * 

o 

0 

B" 

op 

O 

ft 

.ft 

1 

3' 

3 

1 
ft 
P 
o 

o 

-_.  ft 

P 

o 

N| 

< 

5' 

-t                           "i 
_                            a. 

3 
Z- 

5= 

c- 

ft 
en 
o 

3 

o 

5' 

n 

cn" 

ft 

3 

o 

3- 

H- 

o 

a 

£- 

< 

ft 

es  in 
This 

ir. 

c 

ft 

(B 

3" 

H 

3- 
ft 

s                             » 

ef 

5' 

p 

O                                   n 

3^ 

£L 

i 

3 

2. 

n" 

ft* 

5'                          ° 

o 

c 

a 

3                                                         P 

3 

3" 

3 

cid  i 
igiv 

- 

o 

p 

p 

2. 

ft                                        o 

en                                      "I 

= 

3 
— 

3 

o 

pha 

unk 
eart 

•O    3 

- 

- 

> 

3 

3 
o 

3 

c' 
3 
c 
» 
ft 

1  i  iple     pho 
te"  ( mixed  wi 

nown  amount 

hy  phosphate ) 

•a  e  g 

ft      3      to 

n    3" 

B  ,-^ 

2.  5 

3     B 

5' 
3 
o 

- 
p 

s 

r 

n 

B 

| 

0 

E 

3 

5' 

C 

0 
n 

tr 

b' 

X 
p 

3 

o" 

p 
o 

5" 

■    2>Er  .                     3« 

? 

'CHAPTER    XXI. 
URINE:    QUANTITATIVE   ANALYSIS. 

I.     Proteid. 

i.  Scherer's  Coagulation  Method. — The  content  of  coag- 
ulable  proteid  may  be  accurately  determined  as  follows :  Place 
50  c.c.  of  urine  in  a  small  beaker  and  raise  the  temperature 
of  the  fluid  to  about  400  C.  upon  a  water-bath.  Add  dilute 
acetic  acid,  drop  by  drop,  to  the  warm  urine,  to  precipitate 
the  proteid  which  will  separate  in  a  flocculent  form.  Care 
should  be  taken  not  to  add  too  much  acid ;  ordinarily  less  than 
twenty  drops  is  sufficient.  The  temperature  of  the  water  in  the 
water-bath  should  now  be  raised  to  the  boiling-point  and  main- 
tained there  for  a  few  minutes  in  order  to  insure  the  complete 
coagulation  of  the  proteid  present.  Now  filter  the  urine 
through  a  previously  zvashed,  dried  and  zveighed  filter  paper, 
wash  the  precipitated  proteid,  in  turn,  with  hot  water,  95  per 
cent  alcohol  and  with  ether,  and  dry  the  paper  and  precipitate, 
to  constant  weight,  in  an  air-bath  at  no°  C.  Subtract  the 
weight  of  the  filter  paper  from  the  combined  weight  of  the 
paper  and  precipitate  and  calculate  the  percentage  of  proteid 
in  the  urine  specimen. 

Calculation. — To  determine  the  percentage  of  proteid  pres- 
ent in  the  urine  under  examination,  multiply  the  weight  of  the 
precipitate,  expressed  in  grams,  by  2. 

2.  Esbach's  Method. — This  method  depends  upon  the 
precipitation  of  proteid  by  Esbach's  reagent1  and  the  appa- 
ratus used  in  the  estimation  is  Esbach's  albuminometer  (Fig. 
117,  p.  345).  In  making  a  determination  fill  the  albuminometer 
to  the  point  U  with  urine,  then  introduce  the  reagent  until  the 
point  R  is  reached.     Now  stopper  the  tube,  invert  it  slowly 

1  Esbach's   reagent   is   prepared   by   dissolving   10   grams   of  picric   acid 
and  20  grams  of  citric  acid  in  1  liter  of  water. 

344 


urine:  quantitative  analysis. 


345 


several  times  in  order  to  insure  the  thorough  mixing  of  the 
fluids  and  stand  the  tube  aside  for  24  hours.    Creatinin.  resin 
acids,  etc.,  are  precipitated  in  this  method,  and  for  this  and 
other  reasons   it   is   n<>t   as  accurate  as   the 
coagulation   method.      It   is,   however,   ex- 
tensively used  clinically. 

Calculation.- — The  graduations  on  the 
albuminometer  indicate  grams  of  proteid  po- 
liter of  urine.  Thus,  if  the  proteid  precipi- 
tate is  level  with  the  figure  3  of  the  gradu- 
ated scale  this  denotes  that  the  urine  ex- 
amined contains  3  grams  of  proteid  to  the 
liter.  To  express  the  amount  of  proteid  in 
per  cent  simply  move  the  decimal  point  otic 
place  to  the  left.  In  the  case  under  consider- 
ation the  urine  contains  0.3  per  cent  of 
proteid. 

II.     Dextrose. 

1.  Fehling's  Method. — Place  10  c.c.  of 
the  urine  under  examination  in  a  100  c.c. 
volumetric  flask  and  make  the  volume  up  to 
100  c.c.  with  distilled  water.  Thoroughly 
mix  this  diluted  urine,  by  pouring  it  into  a 
beaker  and  stirring  with  a  glass  rod,  then 
transfer  a  portion  of  it  to  a  burette  which  is 
properly  supported  in  a  clamp. 

Xow  place  10  c.c.  of  Fehling's  solution1 
in  a  small  beaker,  dilute  it  with  approxi- 
mately 40  c.c.  of  distilled  water,  heat  to 
boiling,  and  observe  whether  decomposi- 
tion of  the  Fehling's  solution  itself  has 
occurred  as  indicated  by  the  production  of 
a  turbidity.  If  such  turbidity  is  produced  the  Fehling's  solu- 
tion is  unfit  for  use.  Clamp  the  burette  containing  the  diluted 
urine  immediately  over  the  beaker  and  carefully  allow  from  0.5 

1  Directions  for  the  preparation  of  Fehling's  solution  are  given  in  a  note 
at  the  bottom  of  page  8. 


Esbach's  Albumi- 
nometer.   (Ogden.) 


34^  PHYSIOLOGICAL    CHEMISTRY. 

to  i  c.c.  of  the  diluted  urine  to  flow  into  the  boiling  Fehling's 
solution.  Bring  the  solution  to  the  boiling-point  after  each 
addition  of  urine  and  continue  running  in  the  urine  from  the 
burette.  0.5-1  c.c.  at  a  time,  as  indicated,  until  the  Fehling's 
solution  is  completely  reduced,  i.  c,  until  all  the  cupric  oxide 
in  solution  has  been  precipitated  as  cuprous  oxide.  This  point 
will  be  indicated  by  the  absolute  disappearance  of  all  blue  color. 
When  this  end-point  is  reached  note  the  number  of  cubic 
centimeters  of  diluted  urine  used  in  the  process  and  calculate 
the  percentage  of  dextrose  present,  in  the  sample  of  urine 
analyzed,  according  to  the  method  given  on  page  347. 

This  is  a  very  satisfactory  method,  the  main  objection  to 
its  use  being  the  uncertainty  attending  the  determination  of 
the  end-reaction,  i.  e.,  the  difficulty  with  which  the  exact 
point  where  the  blue  color  finally  disappears  is  noted.  Sev- 
eral means  of  accurately  fixing  this  point  have  been  suggested 
but  they  are  practically  all  open  to  objection.  As  good  a 
"  check  "  as  any,  perhaps,  is  to  filter  a  few  drops  of  the  solu- 
tion, through  a  double  paper,  after  the  blue  color  has  appar- 
ently disappeared,  acidify  the  filtrate  with  acetic  acid  and  add 
potassium  ferrocyanide.  If  the  copper  of  the  Fehling's  solu- 
tion has  been  completely  reduced,  there  will  be  no  color  reac- 
tion, whereas  the  production  of  a  brown  color  indicates  the 
presence  of  unreduced  copper.  Harrison  has  recently  sug- 
gested the  following  procedure  to  determine  the  exact  end- 
point:  To  about  1  c.c.  of  a  starch  iodide  solution1  in  a  test- 
tube  add  2-3  drops  of  acetic  acid  and  introduce  into  the 
acidified  mixture  1-2  drops  of  the  solution  to  be  tested.  Un- 
reduced copper  will  be  indicated  by  the  production  of  a  pur- 
plish-red or  blue  color  due  to  the  liberation  of  iodine. 

It  is  ordinarily  customary  to  make  at  least  three  deter- 

1  The  starch-ioclide  solution  may  be  prepared  as  follows:  Mix  0.1  gram 
of  starch  with  cold  water  in  a  mortar  and  pour  the  suspended  starch 
granules  into  75-100  c.c.  of  boiling  water,  stirring  continuously.  Cool  the 
starch  paste,  add  20-25  grams  of  potassium  iodide  and  dilute  the  mixture 
to  250  c.c.  This  solution  deteriorates  upon  standing,  and  therefore  must 
be  freshly  prepared  as  needed. 


URINE:    QUANTITATIVE    ANALYSIS.  347 

miriations  by  Fehling's  method  In-fore  coming  to  a  final 
conclusion  regarding  the  sugar  content  of  the  urine  under 
examination. 

Calculation. — Ten  c.c.  of  Fehling's  solution  is  completely 
reduced  by  0.05  gram  of  dextrose,  [f  y  represents  the  num- 
ber of  cubic  centimeters  of  undiluted  urine  (obtained  by 
dividing  the  burette  reading  by  10)  necessary  to  reduce  the  10 
c.c.  of  Fehling's  solution,  we  have  the  following  proportion: 

v  :  0.05  : :  100 :  .r  ( percentage  of  dextrose) . 

2.  Purdy's  Method. —  I 'inch's  solution1  is  a  modification 
of  Fehling's  solution  and  is  said  to  possess  greater  stability 
than  the  latter.  One  of  the  most  satisfactory  points  about 
the  method  as  suggested  by  Purdy  is  the  ease  with  which  the 
exact  end-reaction  may  be  determined.  In  determining  the 
percentage  of  dextrose  by  this  method  proceed  as  follows: 
Place  35  c.c.  of  Purdy's  solution  in  a  200  c.c.  Erlenmeyer 
flask  and  dilute  the  fluid  with  approximately  two  volumes  of 
distilled  water.  Fit  a  cork,  provided  with  two  perforations, 
to  the  neck  of  the  flask  and  through  one  perforation  introduce 
the  tip  of  a  burette  and  through  the  second  perforation  intro- 
duce a  tube  bent  at  right  angles  in  such  a  manner  as  to  allow 
the  steam  to  escape  and  keep  the  fumes  of  ammonia  away 
from  the  face  of  the  operator  as  completely  as  possible.1     N<  >w 

1  Purdy's  solution  has  the  following  composition : 

Cupric  sulphate    4/52  grams. 

Potassium    hydroxide    23.5      grams. 

Ammonia   (U.  S.  P.,  sp.  gr.  0.9) 3500      c.c. 

Glycerin    38.0      c.c. 

Distilled  water,  to  make  total  volume  1  liter. 

In  preparing  the  solution  bring  the  CuSO<  and  KOH  into  solution  in 
separate  vessels,  mix  the  two  solutions,  cool  the  mixture  and  add  the 
ammonia  and  glycerin.  After  this  has  been  done  the  total  volume  should 
be  made  up  to  1   liter  with  distilled  water. 

Thirty-five  cubic  centimeters  of  Purdy's  solution  is  exactly  reduced  by 
0.02  gram  of  dextrose. 

'This  side  tube  may  also  be  equipped  with  a  simple  air-valve,  thus 
insuring  the  exclusion  of  air  and  thereby  contributing  to  the  accuracy  of 


348  PHYSIOLOGICAL    CHEMISTRY. 

bring  the  solution  to  the  boiling-point  and  add  the  urine,  drop 
by  drop,  until  the  intensity  of  the  blue  color  begins  to  diminish. 
When  this  point  is  reached  add  the  urine  somewhat  more  slozvly 
until  the  blue  color  is  entirely  dissipated  and  an  absolutely 
decolorized  solution  remains.  Take  the  burette  reading  and 
calculate  the  percentage  of  dextrose  in  the  urine  examined 
according  to  the  method  given  below. 

Care  should  be  taken  not  to  boil  the  solution  for  too  long 
a  period,  since,  under  these  conditions,  sufficient  ammonia 
might  be  lost  to  allow  the  cuprous  hydroxide,  CuOH,  to 
precipitate. 

Some  investigators  consider  it  to  be  advisable  to  dilute  the 
urine  before  applying  the  above  manipulation,  but  ordinarily 
this  is  not  necessary  unless  the  urine  has  a  high  content  of 
dextrose  (5  per  cent  or  over).  In  this  event  the  urine  may 
be  diluted  with  2-3  volumes  of  water  and  the  proper  correc- 
tion made  in  the  calculation. 

Calculation. — Thirty-five  c.c.  of  Purdy's  solution  is  com- 
pletely reduced  by  0.02  gram  of  dextrose.  If  y  represents 
the  number  of  cubic  centimeters  of  undiluted  urine  necessary 
to  reduce  35  c.c.  of  Purdy's  solution,  we  have  the  following 
proportion : 

y :  0.02  : :  100 :  x  (percentage  of  dextrose) . 

3.  Fermentation  Method. — This  method  consists  in  the 
measurement  of  the  volume  of  C02  evolved  when  the  dex- 
trose of  the  urine  undergoes  fermentation  with  yeast.  None 
of  the  various  methods  whose  manipulation  is  based  upon  this 
principle  is  absolutely  accurate.  The  method  in  which  Ein- 
horn's  saccharometer  (Fig.  2,  page  10)  is  the  apparatus  em- 
ployed is  perhaps  as  satisfactory  as  any  for  clinical  purposes. 
The  procedure  is  as  follows:  Place  about  15  c.c.  of  urine  in 

the  determination,  inasmuch  as  the  cuprous  salts  would  be  reoxidized 
upon  coming  in  contact  with  the  air.  If  one  is  careful  to  maintain  the 
solution  continuously  at  the  boiling-point  throughout  the  entire  process, 
however,  there  is  no  opportunity  for  air  to  enter  and  therefore  no  need 
of  an  air-valve. 


urine:  quantitative  analysis.  349 

a  mortar,  add  about  1  gram  of  yeast  (tV  of  the  ordinary  cake 
of  compressed  yeasl  I  and  carefully  crush  the  latter  by  means 
of  a  pestle.    Transfer  the  mixture  to  the  saccharometer,  being 

careful  to  note  that  the  graduated  tube  is  completely  tilled  and 
that  no  air  bubbles  gather  at  the  top.  Allow  the  apparatus  to 
stand  in  a  warm  place  (30  C.)  for  1  2  hours  and  Observe  the 
percentage  of  dextrose  as  indicated  by  the  graduated  scale  of 
the  instrument.  Both  the  percentage  of  dextrose  and  the 
number  of  cubic  centimeters  of  CO..  are  indicated  by  the 
graduations  on  the  side  of  the  saccharometer  tube. 

4.  Polariscopic  Examination. — Before  subjecting  urine  to 
a  polariscopic  examination  the  slightly  acid  fluid  should  be 
decolorized  as  thoroughly  as  possible  by  the  addition  of  a 
little  plumbic  acetate.  The  urine  should  be  well  stirred  and 
then  filtered  through  a  filter  paper  which  has  not  been  pre- 
viously moistened.  In  this  way  a  perfectly  clear  and  almost 
colorless  liquid  is  obtained. 

In  determining  dextrose  by  means  of  the  polariscope  it 
should  be  borne  in  mind  that  this  carbohydrate  is  often  accom- 
panied by  other  optically  active  substances,  such  as  proteids. 
la-vulose,  /8-oxybutyric  acid  and  conjugate  glycuronates  which 
may  introduce  an  error  into  the  polariscopic  reading;  the 
method  is,  however,  sufficiently  accurate  for  practical  purposes. 

For  directions  as  to  the  manipulation  of  the  polariscope 

see  page  1 1 . 

III.     Uric  Acid. 

1.  Folin-Shaffer  Method. — Introduce  100  c.c.1  of  urine 
into  a  beaker,  add  25  c.c.  of  the  Folin-Shaffer  reagent2  and 
allow  the  mixture  to  stand.3  without  further  stirring,,  until 
the  precipitate  has  settled   (5-10  minutes).     Filter,  transfer 

'It  is  preferable  to  use  more  than  100  c.c.  of  urine  if  the  fluid  lias  a 
specific  gravity  less  than   1.020. 

2  The  Folin-Shaffer  reagent  consists  of  500  grams  of  ammonium  sul- 
phate. 5  grams  of  uranium  acetate  and  60  c.c.  of  10  per  cent  acetic  acid 
in  650  c.c.  of  distilled  water. 

3  The  mixture  should  not  be  allowed  to  stand  for  too  long  a  time  at 
this  point,  since  uric  acid  may  be  lost  through  precipitation. 


35°  PHYSIOLOGICAL    CHEMISTRY. 

ioo  c.c.  of  the  filtrate  to  a  beaker,  add  5  c.c.  of  concentrated 
ammonia  and  allow  the  mixture  to  stand  for  24  hours. 
Transfer  the  precipitated  ammonium  urate  quantitatively  to 
a  filter  plant,1  using"  10  per  cent  ammonium  sulphate  to 
remove  the  final  traces  of  the  urate  from  the  beaker.  Wash 
the  precipitate  approximately  free  from  chlorides  by  means 
of  10  per  cent  ammonium  sulphate  solution,  remove  the  paper 
from  the  funnel,  open  it  and  by  means  of  hot  water  rinse 
the  precipitate  back  into  the  beaker  in  which  the  urate  was 
originally  precipitated.  The  volume  of  fluid  at  this  point  should 
be  about  100  c.c.  Cool  the  solution  to  room  temperature,  add 
15  c.c.  of  concentrated  sulphuric  acid  and  titrate  at  once  with 
^5-  potassium  permanganate,  K2Mn2Os,  solution.  The  first 
tinge  of  pink  color  which  extends  throughout  the  fluid  after 
the  addition  of  two  drops  of  the  permanganate  solution,  while 
stirring  with  a  glass  rod,  should  be  taken  as  the  end-reaction. 
Take  the  burette  reading  and  compute  the  percentage  of  uric 
acid  present  in  the  urine  under  examination. 

Calculation. — Each  cubic  centimeter  of  ^V  potassium  per- 
manganate solution  is  equivalent  to  3.75  milligrams  (0.00375 
gram)  of  uric  acid.  The  100  c.c.  from  which  the  ammonium 
urate  was  precipitated  is  equivalent  to  only  four-fifths  of  the 
100  c.c.  of  urine  originally  taken,  therefore  we  must  take 
five- fourths  of  the  burette  reading  in  order  to  ascertain  the 
number  of  cubic  centimeters  of  the  permanganate  solution 
required  to  titrate  100  c.c.  of  the  original  urine  to  the  correct 
end-point.  If  y  represents  the  number  of  cubic  centimeters 
of  the  permanaganate  solution  required,  we  may  make  the 
following  calculation : 

y  X  0.00375  =  weight  of  uric  acid  in  100  c.c.  of  urine. 

Calculate  the  quantity  of  uric  acid  in  the  twenty-four  hour 
urine  specimen. 

2.  Heintz  Method. — This  is  a  very  simple  method  and 
was  the  first  one  in  general  use  for  the  quantitative  determi- 

1  The  Schleicher  and  Schiill  hardened  papers  are  the  best  for  this  purpose. 


urine:  quantitative  analysis.  353 

nation  of  uric  acid.  It  is  believed  to  be  somewhat  less  accu- 
rate than  the  method  just  described.  The  procedure  is  as 
follows:  Place  too  c.c.  of  filtered  urine  in  a  beaker,  add  5 
c.c.  of  concentrated  hydrochloric  acid,  stir  the  fluid  thoroughly 

and  stand  it  away  in  a  cool  place  for  -'4  hours.     Filter  off 

the  uric  acid  crystals  upon  a  washed,  dried  and  weighed  filter 
paper  and  wash  them  with  (•<</</  distilled  water,  a  few  cubic 
centimeters  at  a  time  until  the  chlorides  are  removed.  Now 
wash,  in  turn,  with  alcohol  and  with  ether  and  finally  dry  the 
paper  and  crystals  to  constant  weight  at  1  io°  C.  In  the  process 
of  washing  the  uric  acid  free  from  chlorides  an  error  is  intro- 
duced, since  every  cubic  centimeter  of  water  so  used  dissolves 
0.00004  gram  of  uric  acid.  For  this  reason  a  correction  is 
necessary.  It  has  heen  surest ed  that  the  pigment  of  the 
crystals  is  equivalent  in  weight  to  the  .amount  of  uric  acid 
dissolved  by  the  first  30  c.c.  of  water,  and  this  factor  should 
be  taken  into  account  in  the  computation  of  the  percentage  of 
uric  acid. 

Calculation. — Since  100  c.c.  of  urine  was  used  the  cor- 
rected weight  of  the  uric  acid  crystals,  in  grams,  will  express 
the  percentage  of  uric  acid  present. 

IV.    Urea. 

1.  Knop-Hiifner  Hypobromite  Method  (using  Mar- 
shall's Urea  Apparatus). —  Place  the  thumb  over  the  side 
opening  of  the  bulbed-tube  of  the  apparatus  (  Fig.  1 18,  p.  352  ) 
and  carefully  fill  the  tube  with  sodium  hypobromite  solution.1 
(lose  the  opening  in  the  end  of  the  tube  with  a  rubber 
stopper,    incline    the    tube    to    allow    air-bubbles    to    escape 

1  The  ingredients  of  the  sodium  hypobromite  solution  should  be  pre- 
pared in  the  form  of  two  separate  solutions.  When  needed  for  use  mix 
equal  volumes  of  solution  a,  solution  6  and  water. 

(a)  Dissolve  125  grams  of  sodium  bromide  in  water,  add  125  grams  of 
bromine  and  make  the  total  volume  of  the  solution  1   liter. 

(b)  A  solution  of  sodium  hydroxide  having  a  specific  gravity  of  T.250. 
This  is  approximately  a  22.5  per  cent  solution. 

Preserve  both  solutions  in  rubber-stoppered  bottles. 


352 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  i  i  8. 


and  finally  invert  the  tube  and  fix  the  stoppered  end  in 
the  saucer-shaped  vessel.  By  means  of  the  graduated  pi- 
pette rapidly  introduce  I  c.c.  of  urine1  into  the  hypobromite 

solution  through  the  side  open- 
ing of  the  bulbed-tube.  With- 
draw the  pipette  immediately 
after  the  urine  has  been  intro- 
duced. When  the  decomposi- 
tion of  the  urea  is  completed 
(10-20  minutes)  gently  tap 
the  bulbed-tube  with  the  finger 
in  order  to  dislodge  any  gas-bub- 
bles which  may  have  collected 
on  the  inner  surface  of  the  glass. 
The  atmospheric  pressure  should 
now  be  equalized  by  attach- 
ing the  funnel-tube  to  the 
bulbed-tube  at  the  side  opening 
and  introducing  hypobromite 
solution  into  it  until  the  columns 
of  liquid  in  the  two  tubes  are 
uniform  in  height.  The  gradu- 
ated scale  of  the  bulbed-tube 
should  now  be  read  in  order 
to  determine  the  number  of 
cubic  centimeters  of  nitrogen  gas  evolved.  By  means  of  the 
appended  formula  the  weight  of  the  urea  present  in  the  urine 
under  examination  may  be  computed. 

Calculation.2 — By  properly  substituting  in  the  following 
formula  the  weight  of  urea,  in  grams,  contained  in  the  volume 
of  urine  decomposed  ( 1  c.c.  or  more)  may  readily  be  deter- 
mined : 


1  ■■•;■  'lli'l'  I1  ' 


'iliMiiilmili- 


Marshall's    Urea   Apparatus. 
(Tyson.) 

a,  Bulbed  measuring  tube ;  b, 
saucer-shaped  vessel ;  c,  graduated 
pipette  :   d,  funnel-tube. 


1  Ordinarily  I  c.c.  of  urine  is  sufficient ;  more  may  be  used,  however,  if 
its  content  of  urea  is  very  low. 

2  0.003665  =  coefficient  of  expansion  of  gases  for  i°  C.     354.5  =  number 
of  c.c.  of  nitrogen  gas  evolved  from  1  gram  of  urea. 


w 


trim::    QUANTITATIVE    ANALYSIS.  353 


354-5  +  760(1  +0.003665O 

w      weight  of  urea,  in  grams. 

v  =  :'observed  volume  of  nitrogen  expressed  in  cubic  centi- 
meters. 

p  =  barometric  pressure  expressed  in  mm.  of  mercury. 

T=  tension  of  aqueous  vapor1  for  temperature  /. 

/        temperature  (centigrade). 

1 1  we  wish  to  calculate  the  percentage  of  urea  we  may  do  so 
by  means  of  the  following-  proportion  in  which  y  represents  the 
volume  of  urine  used  and  w  denotes  the  weight  of  the  urea  con- 
tained in  the  volume  y  : 

v  :  w : :  100 :  x  (percentage  of  urea) . 

Sodium  hypobromite  solution  may  also  be  employed  for  the 
determination  of  urea  in  the  apparatus  devised  by  Hiifner 
which  is  pictured  in  Fig.  1  [9,  page  354. 

2.  Knop-Hiifner  Hypobromite  Method  (using  the 
Doremus-Hinds  Ureometer). — In  common  with  the  method 
already  described  this  method  depends  upon  the  meas- 
urement of  the  volume  of  nitrogen  gas  liberated  when  the 
urea  of  the  urine  is  decomposed  by  means  of  sodium  hypobro- 
mite solution.  The  Doremus-Hinds  ureometer  (Fig.  120,  p. 
355),  is  one  of  the  simplest  and  cheapest  forms  of  apparatus  in 
general  use  for  the  determination  of  urea  by  the  hypobromite 
process.  In  using  this  apparatus  proceed  as  follows :  Fill  the 
side  tube  B  and  the  lumen  of  the  stopcock  C  with  the  urine 

1  The  values  of  T  for  the  temperatures  ordinarily  met  with  are  given 
in  the  following  table : 

Temp.                    Tension  Temp.                    Tension 
in    mm.                                                          in    mm. 

15°  C 12.677  210  C 18.505 

160  C 13519  220  C 19675 

170  C 14.009  23°  C 20.909 

180  C 15.351  240  C 22.211 

19°  C 16.345  250  C 23582 

20°    C I7-396 

24 


354 


PHYSIOLOGICAL    CHEMISTRY, 


under  examination.  Carefully  wash  out  tube  A  with  water 
and  introduce  into  it  sodium  hypobromite  solution1  being  care- 
ful to  fill  the  bulb  sufficiently  full  to  prevent  the  entrance  of  air 

into  the  graduated  portion. 


Fig.  119. 


Hufxer's  Urea  Apparatus 


Now  allow  i  c.c.  of  urine2 
to  flow  from  tube  B  into 
tube  A  and  after  the  evo- 
lution of  gas  bubbles  has 
ceased  (10-20  minutes)  take 
the  reading  of  the  graduated 
scale  on  tube  A. 

In  common  with  all  other 
methods  which  are  based 
upon  the  decomposition  of 
urea  by  means  of  hypobro- 
mite solution,  this  method 
is  not  absolutely  correct.  It 
is,  however,  sufficiently  ac- 
curate for  ordinary  clinical 
purposes. 

Calculation. — Observe  the 
reading  on  the  graduated 
scale  of  tube  A.  This  tube 
is  so  graduated  as  to  rep- 
resent the  weight  of  urea, 
in  grams,  per  cubic  centi- 
meter of  urine.  If  we  wish 
to  compute  the  percentage 
of  urea  present  this  may  be 
done  very  readily  by  simply 
moving  the  decimal  point  two 
if  the  reading  is  0.02  gram  the  urine 


places  to  the  right,  e.  g., 
contains  2  per  cent  of  urea. 

1  For  directions  as  to  the  preparation  of  this  solution  see  page  351. 

'  If  the  content  of  urea  in  the  urine  under  examination  is  large,  the 
urine  may  be  diluted  with  water  before  determining  the  urea.  If  this  is 
done  it  must  of  course  be  taken  into  consideration  in  computing  the  con- 
tent of  urea. 


urine:  quantitative  analysis. 


3  55 


3.  Folin's  Method.-  This  is  one  of  the  most  accurate 
methods  vet  devised  for  the  determination  of  urea  in  the  urine. 
The  procedure  is  as  follows:  Place  5  c.c.  of  urine  in  a  200 
Erlenmeyer  flask  and  add  to  it 
5  c.c.  of  concentrated  hydro 
chloric  acid.  20  grams  "I  crys- 
tallized magnesium  chloride,  a 
piece  of  paraffin  the  size  of  a 
hazel  nut  and  _•  3  drops  of  a  1 
per  cent  aqueous  solution,  of 
"alizarin  red."  Tnsert  a  Folin 
safety  tube  (Fig.  121, p.  356)  in- 
to the  neck  of  the  flask  and  boil 
the  mixture  until  each  drop  of 
reflow  from  the  safety  tube  pro- 
duces a  very  perceptible  bump; 
the  heat  is  then  reduced  some- 
what and  continued  one  hour.1- 
The  contents  of  the  flask  must 
not  remain  alkaline  and  to  ob- 
viate this,  at  the  first  appearance 
of  a  reddish  tinge  in  the  contents 
of  the  flask  a  fez*.'  drops  of  the 
acid  distillate  are  shaken  back 
into  the  flask.  At  the  end  of  an 
hour  the  contents  of  the  vessel 
are  transferred  to  a  1  liter  flask 
with  about  700  c.c.  of  distilled  water,  about  20  c.c.  of  10 
per  cent  potassium  hydroxide  or  sodium  hydroxide  solution 
i-  added  and  the  mixture  distilled  into  a  known  volume  of  ft 
sulphuric  acid  until  the  contents  of  the  flask  are  nearly  dry 
or  until  the  distillate  fails  to  give  an  alkaline  reaction  to 
litmus,  showing  the  absence  of  ammonia.  The  time  devoted 
to  this  process  is  ordinarily  about  an  hour.     Boil  the  distillate 


DOREMUS-HlNDS     UrEOMETER. 


1  If  low  results  are  obtained  the  heating  should  be  continued  one  and  one- 
half  hours  in  subsequent  determinations. 


356 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  121. 


osH 


a  few  moments  to  free  it  from  COo,  then  cool  and  titrate  the 
mixture  with  y-g-  sodium  hydroxide,  using  "  alizarin  red  "  as 
indicator. 

A  "  check  "  experiment  should  always  be  made  to  determine 
the  original  ammonia  content  of  the  urine  and  of  the  magne- 
sium chloride,  if  it  is  not  ab- 
solutely pure,  which  of  course 
should  be  subtracted  from  the 
total  amount  of  ammonia  as 
determined  by  the  above  process. 
The  Folin  method  is  extremely 
accurate  under  all  conditions 
creep t  when  the  urine  contains 
sugar.  When  this  is  the  case 
the  carbohydrate  and  the  urea 
unite,  upon  being  heated,  and 
form  a  very  stable  combination. 
For  this  reason  the  Folin  method 
is  not  suitable  for  use  in  the 
examination  of  such  urines. 
The  best  method  for  use  under 
such  conditions  is  the  combi- 
nation M6rner-Sjoqvist-F  o  1  i  n 
method  which  is  given  below. 
4.  Morner  -  Sjoqvist  -  Folin 
Method. — As  has  already  been 
stated  in  the  last  experiment 
this  method  excels  the  Folin 
method  in  accuracy  only  in  the 
determination  of  urea  in  the 
presence  of  carbohydrate  bodies.  Briefly  the  procedure  is  as 
follows:1  Bring  the  major  portion  of  1.5  grams  of  powdered 
barium  hydroxide  into  solution  in  5  c.c.  of  urine  in  a  small 

1  The  original  description  of  the  method  may  be  found  in  an  article  by 
Morner:  Skandinavisches  Archiv  fur  Physiologic,   1903,  xiv,  p.  297. 


Folin's   Urea  Apparatus. 


urine:  quantitative  analysis.  357 

flask,  and  treat  the  mixture  with  rooc.c.  of  an  alcohol-ether  so- 
lution, consisting  of  two  volumes  of  97  per  cent  alcohol  and  one 
volume  of  ether.  Stopper  the  flask  and  allow  it  to  stand  12-24 
hours.  Filter  off  the  precipitate,  wash  it  with  the  alcohol-ether 
mixture  and  remove  the  alcohol  and  ether  from  the  filtrate  by 
distillation,  being  careful  to  keep  the  temperature  of  the  mix- 
ture below  50  l".1  Treat  the  remaining  fluid  (about  25  c.c.) 
with  2  c.c.  of  hydrochloric  acid  i  sp.  gr.  [.124)  transfer  it  care- 
fully to  a  200  c.c.  flask  and  evaporate  the  mixture  to  dryness  on 
a  water-bath.  Now  add  20  grams  of  crystallized  magnesium 
chloride  and  2  C.C.  of  concentrated  hydrochloric  acid  to  the 
residue  and  after  fitting  the  flask  with  a  return  cooler  boil  the 
mixture  on  a  wire  gauze  over  a  small  flame  for  two  hours 
Cool  the  solution,  dilute  to  750  c.c.  or  to  1000  c.c.  with  water, 
render  the  mixture  alkaline  with  potassium  hydroxide  or 
sodium  hydroxide,  distil  off  the  ammonia  and  collect  it  in  an 
acid  solution  of  known  strength.  Boil  the  distillate  to  remove 
carh.  >n  dioxide,  cool  and  titrate  with  an  alkali  of  known 
strength.  In  this  method,  as  well  as  in  Folin's  method  (see 
p.  355),  correction  must  he  made  for  the  ammonia  originally 
present  in  the  urine  and  in  the  magnesium  chloride. 

V.  Ammonia. 
1.  Folin's  Method. — Place  2^  c.c.  of  urine  in  an  rerometer 
cylinder,  30-45  cm.  in  height  (Fig  i_'_\  p.  358),  add  about  one 
gram  of  dry  sodium  carbonate  and  introduce  some  crude 
petroleum  to  prevent  foaming-.  Insert  into  the  neck  of  the 
cylinder  a  rubber  stopper  provided  with  two  perforations  into 
each  of  which  passes  a  glass  tube  one  of  which  reaches  below 
the  surface  of  the  liquid.  The  shorter  tube  ( 10  cm.  in  length  | 
is  connected  with  a  calcium  chloride  tube  filled  with  cotton 
and  this  tube  is  in  turn  joined  to  a  glass  tube  extending 
to  the  bottom  of  a  500  c.c.  wide  mouthed  ilad<  which  is 
intended  to  absorb  the  ammonia. and  for  this  purpose  should 
contain   20   c.c.    of   y$    sulphuric    acid,    200   c.c.    of    distilled 

1  There  is  some  decomposition  of  urea  at  6o°  C. 


35* 


PHYSIOLOGICAL    CHEMISTRY. 


water  and  a  few  drops  of  an  indicator  ("  alizarin  red  ").     To 
insure  the  complete  absorption  of  the  ammonia  the  absorption 


Fig.  122. 


Folin's  Ammonia  Apparatus. 

flask  is  provided  with  a  Folin  absorption  tube  (Fig.  123,  p.  359) 
which  is  very  effective  in  causing  the  air  passing  from  the 
cylinder  to  come  into  intimate  contact  with  the  acid  in  the 
absorption  flask.  In  order  to  exclude  any  error  due  to  the 
presence  of  ammonia  in  the  air  a  similar  absorption  apparatus 
to  the  one  just  described  is  attached  to  the  other  side  of  the 
aerometer  cylinder,  thus  insuring  the  passage  of  ammonia-free 
air  into  the  cylinder.  With  an  ordinary  filter  pump  and  good 
water  pressure  the  last  trace  of  ammonia  should  be  removed 
from  the  cylinder  in  about  one  and  one-half  hours.1  The 
number  of  cubic  centimeters  of  the  jq  sulphuric  acid  neutral- 
ized by  the  ammonia  of  the  urine  may  be  determined  by  direct 
titration  with  y^   sodium  hydroxide. 

This  is  one  of  the  most  satisfactory  methods  yet  devised  for 
the  determination  of  ammonia. 

1  With  any  given  filter  pump  a  A  check  "  test  should  be  made  with  urine 
or  better  with  a  solution  of  an  ammonium  salt  of  known  strength  to  de- 
termine how  long  the  air  current  must  be  maintained  to  remove  all  the 
ammonia  from  2;  c.c.  of  the  solution. 


urine:    QUANTITATIVE   ANALYSIS. 


359 


Fig.  123. 


Calculation. — Subtract  the  number  of  cubic  centimeters  of 
idium  hydroxide  used  in  the  titration  from  the  number  of 
cubic  centimeters  of  ,Nn  sulphuric  acid  taken.  The  remainder 
is  the  number  of  cubic  centimeters  of 
sulphuric  acid  neutralized  by  the  -\7/:.  of 
the  urine.  1  cc.  of  n!  sulphuric  acid  is 
equivalent  to  0.0017  gram  of  Ar//.:.  There- 
it  y  represents  the  volume  of  urine 
used  in  the  determination  and  y'  the  num- 
ber of  cubic  centimeters  of  fV  sulphuric 
acid  neutralized  by  the  A7/..  af  the  urine. 
we  have  the  following  proportion: 

y :  100 : :  v'  X  0.00 1 7  :  x  (percentage  of  X 1 1 .. 
in  the  urine  examined). 


Calculate    the    quantity    of    XH.    in    the 
twenty-four  hour  urine  specimen. 


VI.     Nitrogen. 

Kjeldahl  Method.1  —  The  principle  of 
this  method  is  the  conversion  of  the  various 
nitrogenous  bodies  of  the  urine  into  ammonium  sulphate  by 
boiling  with  concentrated  sulphuric  acid,  the  subsequent  de- 
composition of  the  ammonium  sulphate  by  means  of  a  fixed 
alkali  (XaOH)  and  the  collection  of  the  liberated  ammonia 
in  an  acid  of  known  strength.  Finally,  this  partly  neutralized 
acid  solution  is  titrated  with  an  alkali  of  known  strength  and 
the  nitrogen  content  of  the  urine  under  examination  com- 
puted. 

The  procedure  is  as  follows :  Place  5  cc.  of  urine  in  a 
200-300  cc.  long-necked,  Jena  glass  Kjeldahl  digestion  flask, 
add  20  cc  of  concentrated  sulphuric  acid  and  about  0.2  gram 
of  cupric  sulphate  and  boil  the  mixture  for  some  time  after 

1  There  are  numerous  modifications  of  the  original  Kjeldahl  method; 
the  one  described  here,  however,  has  given  excellent  satisfaction  and  is 
recommended  for  the  determination  of  the  nitrogen  content  of  urine. 


Folin     Absorption 
Tube. 


360  PHYSIOLOGICAL    CHEMISTRY. 

it  is  colorless  (about  one  hour).  Allow  the  flask  to  cool  and 
transfer1  the  contents,  by  means  of  about  200  c.c.  of  water, 
to  a  750  c.c.  Jena  glass  distillation  flask.  Add  a  little  more 
of  a  concentrated  solution  of  NaOH  than  is  necessary 
to  neutralize  the  sulphuric  acid2  and  introduce  into  the  flask 
a  little  coarse  pumice  stone  or  a  few  pieces  of  granulated 
zinc,  to  prevent  bumping,  and  a  small  piece  of  paraffin  to 
lessen  the  tendency  to  froth.  By  means  of  a  safety-tube 
connect  the  flask  with  a  condenser  so  arranged  that  the  de- 
livery-tube passes  into  a  vessel  containing  a  known  volume 
(the  volume  used  depending  upon  the  nitrogen  content  of 
the  urine)  of  y\  sulphuric  acid,  using  care  that  the  end  of 
the  delivery-tube  reaches  beneath  the  surface  of  the  fluid.3 
Mix  the  contents  of  the  distillation  flask  very  thoroughly  by 
shaking  and  distil  the  mixture  until  its  volume  has  diminished 
about  one-half.  Titrate  the  partly  neutralized  y\  sulphuric 
acid  solution  by  means  of  y\  sodium  hydroxide,  using  congo 
red  as  indicator,  and  calculate  the  content  of  nitrogen  of  the 
urine  examined. 

Calculation. — Subtract  the  number  of  cubic  centimeters 
of  yq  sodium  hydroxide  used  in  the  titration  from  the  number 
of  cubic  centimeters  of  y$  sulphuric  acid  taken.  The  re- 
mainder is  equivalent  to  the.  number  of  cubic  centimeters  of  yv 
sulphuric  acid,  neutralized  by  the  ammonia  of  the  urine. 
One  c.c.  of  ^  sulphuric  acid  is  equivalent  to  0.0014  gram  of 
nitrogen.  Therefore,  if  y  represents  the  volume  of  urine  used 
in  the  determination,  and  y'  the  number  of  cubic  centimeters  of 
Yq  sulphuric  acid  neutralized  by  the  ammonia  of  the  urine,  we 
have  the  following  proportion : 

'A  very  satisfactory  modification  of  this  procedure  includes  the  use  of  a 
750  c.c.  flask  for  both  the  digestion  and  the  distillation,  thus  making  unnec- 
essary any  transfer  of  contents. 

2  This  concentrated  sodium  hydroxide  solution  should  be  prepared  in 
quantity  and  "  check  "  tests  made  to  determine  the  volume  of  the  solution 
necessary  to  neutralize  the  volume  (20  c.c.)  of  concentrated  sulphuric 
acid  used. 

3 This  delivery-tube  should  be  of  large  caliber  in  order  to  avoid  the 
"sucking  back"  of  the  fluid. 


urine:  quantitative  analysis.  361 

y:  100: :  y'  X  0.0014 \x  (percentage  of  nitrogen  in  the  urine 

examined ). 

Calculate  the  quantity  of  nitrogen  in  the  twenty-four  hour 

urine  specimen. 

VII.     Sulphur. 

1.  Total  Sulphates. — Folin' s  Method. — Place  25  c.c.  of 
urine  in  a  200  -'50  c.c.  Erlenmeyer  flask,  add  20  c.c.  of  dilute 
hydrochloric  acid1  (1  volume  of  concentrated  11(1  to  4  vol- 
umes of  water)  and  gently  boil  for  20  30  minutes.  To  mini- 
mize the  loss  of  water  by  evaporation  the  mouth  of  the  flask 
should  be  covered  with  a  small  watch  glass  during  the  boiling 
process.  Cool  the  tlask  for  2-3  minutes  in  running  water,  and 
dilute  the  contents  to  about  150  c.c.  by  means  of  cold  water. 
Add  ro  c.c.  of  a  5  per  cent  solution  of  barium  chloride  slowly. 
drop  by  drop,  to  the  cold  solution.2  The  contents  of  the  flask 
should  not  be  stirred  or  shaken  during  the  addition  of  the 
barium  chloride.  Allow  the  mixture  to  stand  at  least  one 
hour,  then  shake  up  the  solution  and  filter  it  through  a 
weighed  Gooch  crucible.3 

Wash  the  precipitate  of  BaSO,  with  about  250  c.c.  of  cold 
water,  dry  it  in  an  air-bath  or  over  a  very  low  flame,  then 
ignite.  '  a  m  >1  and  weigh. 

1  If  it  is  desired,  50  c.c.  of  urine  and  4  c.c.  of  concentrated  acid  may  be 
used  instead. 

"A  dropper  or  capillary  funnel  made  from  an  ordinary  calcium  chloride 
tube  and  so  constructed  as  to  deliver  10  c.c.  in  2-3  minutes  is  recom- 
mended for  use  in  adding  the  barium  chloride. 

3  If  a  Gooch  crucible"  is  not  available  the  precipitate  of  BaSOi  may  be 
filtered  off  upon  a  washed  filter  paper  (Schleicher  &  Schtill's,  Xo.  589,  blue 
ribbon)  and  after  washing  the  precipitate  with  about  250  c.c.  of  cold 
water  the  paper  and  precipitate  may  be  dried  in  an  air-bath,  or  over  a 
low  flame.  The  ignition  may  then  be  carried  out  in  the  usual  way  in  the 
ordinary  platinum  or  porcelain  crucible.  In  this  case  correction  must  be 
made  for  the  weight  of  the  ash  of  the  filter  paper  used. 

*  Care  must  be  taken  in  the  ignition  of  precipitates  in  Gooch  crucibles. 
The  flame  should  never  be  applied  directly  to  the  perforated  bottom  or  to 
the  sides  of  the  crucible,  since  such  manipulation  is  invariably  attended  by 
mechanical  losses.  The  crucibles  should  always  be  provided  with  lids  and 
tight    bottoms   during    the    ignition.     In    case    porcelain    Gooch    crucibles, 


362  PHYSIOLOGICAL    CHEMISTRY. 

Calculation. — Subtract  the  weight  of  the  Gooch  crucible 
from  the  weight  of  the  crucible  and  the  BaS04  precipitate 
to  obtain  the  weight  of  the  precipitate.  The  weight  of  SO31 
in  the  volume  of  urine  taken  may  be  determined  by  means  of 
the  following  proportion : 

Mol.  wt.         Wt.  of        Mol.  Wt. 

BaS04 :  BaS04 : :  S03 :  x  (wt.  of  S03  in  grams), 
ppt. 

Representing  the  weight  of  the  BaS04  precipitate  by  3'  and 
substituting  the  proper  molecular  weights,  we  have  the  follow- 
ing proportion : 

231.7:  3' ::  79.5  :  x  (wt.   of  SOs  in  grams  in  the  quantity  of 

urine  used). 

Calculate  the  quantity  of  S03  in  the  twenty-four  hour 
specimen  of  urine. 

To  express  the  result  in  percentage  of  S03  simply  divide 
the  value  of  x,  as  just  determined,  by  the  quantity  of  urine 
used. 

2.  Inorganic  Sulphates. — Folin's  Method. — Place  25  c.c. 
of  urine  and  100  c.c.  of  water  in  a  200-250  c.c.  Erlenmeyer 
flask  and  acidify  the  diluted  urine  with  10  c.c.  of  dilute  hydro- 
chloric acid  (1  volume  of  concentrated  HC1  to  4  volumes  of 
water).  In  case  the  urine  is  dilute  50  c.c.  may  be  used  instead 
of  25  c.c.  and  the  volume  of  water  reduced  proportionately. 
From  this  point  proceed  as  indicated  in  the  method  for  the 
determination  of  Total  Sulphates,  page  361. 

Calculate  the  quantity  of  inorganic  sulphates,  expressed  as 
S03,  in  the  twenty-four  hour  urine  specimen. 

whose  bottoms  are  not  provided  with  a  non-perforated  cap,  are  used,  the 
crucible  may  be  placed  upon  the  lid  of  an  ordinary  platinum  crucible 
during  ignition.  The  lid  should  be  supported  on  a  triangle,  the  crucible 
placed  upon  the  lid  and  the  flame  applied  to  the  improvised  bottom.  Igni- 
tion should  be  complete  in  10  minutes  if  no  organic  matter  is  present. 

1  It  is  considered  preferable  by  many  investigators  to  express  all  sulphur 
values  in  terms  of  S  rather  than  SO3. 


urine:  quantitative  analysis.  363 

Calculation. — Calculate  according  to  the  directions  given 
under  Total  Sulphates,  page  36] . 

3.  Ethereal  Sulphates.— /•'.  <//'//  V  Method.  Place  [25  c.c. 
of  urine  in  an  Erlenmeyer  flask  of  suitable  size,  dilute  it  with 
75  c.c.  of  water  and  acidify  the  mixture  with  30  c.c.  of  dilute 
hydrochloric  acid  (  1  volume  of  concentrated  Mel  to  4  vol- 
umes of  water).  To  the  cold  solution  add  20  c.c.  of  a  5  per 
cent  solution  of  barium  chloride,  drop  by  drop.1  Allow  the 
mixture  to  -tand  about  one  hour  then  filter  it  through  a  dry 
filter  paper.2  Colled  [25  c.c.  of  the  filtrate  and  boil  it  gently 
for  at  least  one-half  hour.  Cool  the  solution,  filter  off  the 
precipitate  of  BaSO.,.  wash  and  ignite  it  according  to  the 
directions  given  on  page  36] . 

Calculation. — The  weight  of  the  BaS04  precipitate  should 
be  multiplied  by  2  since  only  one-half  (125  c.c.)  of  the  total 
volume  (250  c.c.)  of  fluid  was  precipitated  by  the  barium 
chloride.  The  remaining  calculation  should  he  made  accord- 
ing to  directions  given  under  Total  Sulphates,  page  361. 

Calculate  the  quantity  of  ethereal  sulphates,  expressed  as 
S03,  in  the  twenty-four  hour  urine  specimen. 

4.  Total  Sulphur. — Osborne-Folin Method. — Place  25  c.c. 
of  urine3  in  a  200-250  c.c.  nickel  crucible  and  add  about  3 
grams  of  sodium  peroxide.  Evaporate  the  mixture  to  a  syrup 
and  heat  it  carefully  until  it  solidifies  (15  minutes).  Now 
remove  the  crucible  from  the  flame  and  allow  it  to  cool. 
Moisten  the  residue  with  1-2  c.c.  of  water,1  sprinkle  about. 
7-8  grams  of  sodium  peroxide  over  the  contents  of  the  cru- 
cible and   fuse  the  mass   for  about    10  minutes.     Allow   the 

1  Set'  note   (2)   at  the  bottom  of  page  361. 

'  This  precipitate  consists  of  the  inorganic  sulphates.  If  it  is  desired, 
this  BaSOi  precipitate  may  be  collected  in  a  Gooch  crucible  or  on  an 
ordinary  quantitative  filter  paper  and  a  determination  of  inorganic  sul- 
phates made,  using  the  same  technique  as  that  suggested  on  p.  361.  In 
this  way  we  are  enabled  to  determine  the  inorganic  and  ethereal  sulphates 
in  the  same  sample  of  urine. 

3  If  the  urine  is  very  dilute  50  c.c.  may  be  used. 

*  This  moistening  of  the  residue  with  a  small  amount  of  water  is  very 
essential  and  should  not  be  neglected. 


364  PHYSIOLOGICAL    CHEMISTRY. 

crucible  to  cool  for  a  few  minutes,  add  about  100  c.c.  of  water 
to  the  contents  and  heat  at  least  one-half  hour,  to  dissolve 
the  alkali  and  decompose  the  sodium  peroxide.  Next  rinse 
the  mixture  into  a  400-456  c.c.  Erlenmeyer  flask,  by  means 
of  hot  water,  and  dilute  it  to  about  250  c.c.  Heat  the  solu- 
tion nearly  to  the  boiling-point  and  add  concentrated  hydro- 
chloric acid  slowly  until  the  nickelic  oxide,  derived  from  the 
crucible,  is  just  brought  into  solution.1  A  few  minutes  boiling 
should  now  yield  a  clear  solution.  In  case  too  little  peroxide 
or  too  much  water  was  added  for  the  final  fusion  a  clear 
solution  will  not  be  obtained.  In  this  event  cool  the  solution 
and  remove  the  insoluble  matter  by  filtration. 

To  the  clear  solution  add  5  c.c.  of  very  dilute  alcohol  (about 
18-20  per  cent)  and  continue  the  boiling  for  a  few  minutes. 
The  alcohol  is  added  to  remove  the  chlorine  which  was  formed 
when  the  solution  was  acidified.  Add  10  c.c.  of  a  10  per  cent 
solution  of  barium  chloride,  slowly,  drop  by  drop,2  to  the 
liquid.  Allow  the  precipitated  solution  to  stand  in  the  cold 
two  days  and  then  filter  and  continue  the  manipulation  accord- 
ing to  the  directions  given  under  Total  Sulphates,  page  361. 

Calculation. — Make  the  calculation  according  to  directions 
given  under  Total  Sulphates,  p.  361.  Calculate  the  quantity 
of  sulphur,  expressed  as  SOs  or  S,  present  in  the  twenty-four 
hour  urine  specimen. 

5.  Total  Sulphur. — Sodium  Hydroxide  and  Potassium 
Nitrate  Fusion  Method. — Place  25  c.c.  of  urine  in  a  silver 
crucible  and  evaporate  to  a  thick  syrup  on  a  water-bath.  Add 
10  grams  of  sodium  hydroxide  and  2  grams  of  potassium 
nitrate  to  the  residue  and  fuse  the  mass,  over  an  alcohol  flame, 
until  all  organic  matter  has  disappeared  and  the  fused  mixture 
is  clear.  Cool  the  mixture,  transfer  it  to  a  casserole,  by  means 
of  hot  water,  acidify  slightly  with  hydrochloric  acid  and  evap- 
orate it  to  dryness  on  a  water-bath.  Moisten  the  residue 
with  a  few  drops  of  dilute  hydrochloric  acid  and  bring  it  into 

1  About  18  c.c.  of  acid  is  required  for  8  grams  of  sodium  peroxide. 
:  See  note    (2)    at   the  bottom  of  page  361. 


urine:  quantitative  analysis.  365 

solution  with  hoi  water.  Filter,  heal  the  filtrate  to  boiling 
.■Hid  immediately  precipitate  it  by  the  addition  of  to  c.c.  of  a 
[O  per  rent  solution  of  barium  chloride,  adding  the  solution 
slowly,  drop  by  drop.  Allow  the  precipitated  solution  to 
stand  2  hours  and  filter  while  cold,  [gnite,  weigh  and  calcu- 
late acc<  »rding  todirecti*  »ns  given  under  Ti  ital  Sulphates,  ]».  361. 

Compute  the  quantity  of  sulphur,  expressed  as  S03  or  S, 
present  in  the  twenty-four  hour  urine  specimen. 

6.  Total  Sulphur.—  Sherman's  Compressed  Oxygen 
Method.1  Evaporate  as  much  urine  on  an  absorbent  filter 
block2  at  60 °  C.  as  the  block  will  conveniently  absorb  and  burn 
the  block  so  prepared  in  a  bomb-calorimeter8  using  25-30 
atmospheres  of  oxygen.  Connect  the  bomb  with  a  wash-bottle 
containing  water,  and  allow  the  gas  to  bubble  through  the 
liquid  until  the  high  pressure  within  the  apparatus  has  been 
reduced  to  atmospheric  pressure.  Xow  open  the  bomb  and 
thoroughly  rinse  the  interior,  using  water  from  the  wash- 
bottle  for  the  first  rinsing.  Dissolve  any  ash  found  in  the  com- 
bustion capsule  in  hydrochloric  acid  and  add  this  solution  to  the 
main  solution.  Evaporate  to  150  c.c,  filter  and  cool  the  filtrate. 
Add  10  c.c.  of  a  5  per  cent  solution  of  barium  chloride  to  the 
cold  filtrate,  slowly,  drop  by  drop.'1  The  contents  of  the  flask 
should  not  be  stirred  or  shaken  during  the  addition  of  the 
barium  chl<  >ride.  Allow  the  mixture  to  stand  at  least  one  hour, 
then  shake  up  the  solution  and  filter  it  through  a  weighed 
Gooch  crucible.  Manipulate  the  precipitate  of  BaS04  accord- 
ing to  directions  given  under  Total  Sulphates,  page  361. 

Calculate  the  quantity  of  sulphur,  expressed  as  S03  or  S, 
present  in  the  twenty-four  hour  urine  specimen. 

1  See  Sherman's  Organic  Analysis,  p.  19. 

2  Only  a  small  amount  of  urine  should  he  added  at  one  time,  it  being 
necessary  to  make  several  evaporations  before  the  block  contains  sufficient 
urinary  residue  to  proceed  with  the  combustion. 

3  The  Berthelot-Atwater  apparatus  (Fig.  124,  page  366)  is  well  adapted 
to  this  purpose. 

4  See  note   (2)   at  the  bottom  of  page  361. 


366 


PHYSIOLOGICAL    CHEMISTRY. 
Fig.  124. 


Berthelot-Atwater   Bomb   Calorimeter.        (Cross-sectiox   of  Apparatus 

as  Ready  for  Use.) 
A,  Steel  cup  or  bomb  proper ;  C,  collar  of  steel ;  G,  opening  through  which 
oxygen  is  forced  into  the  bomb  ;  H  and  I',  insulated  wires  which  serve  to  conduct 
an  electric  current  for  igniting  the  substance  which  is  held  in  the  small  capsule; 
L,  a  stirrer  which  serves  to  keep  the  water  surrounding  the  bomb  in  motion  and 
insures  the  equalization  of  temperature ;  P,  a  delicate  thermometer  which  shows 
the  rise  in  temperature  of  the  water  surrounding  the  bomb. 


urine:  quantitative  analysis.  v  7 

VIII.    Phosphorus. 

i.  Total  Phosphates. — Uranium  Acetate  Method. — To  50 
c.c.  of  urine  in  a  small  beaker  or  Erlenmeyer  flask  add  5  c.c.  of 
a  special  sodium  acetate  solution1  and  heat  the  mixture  to  the 
boiling-point.  From  a  burette,  run  into  the  hot  mixture,  drop 
by  drop,  a  standard  solution  of  uranium  acetate2  until  a  precipi- 
tate ceases  t<>  form  and  a  drop  of  the  mixture  when  removed 
by  means  of  a*  glass  rod  and  brought  in  contact  with  a  drop 
■  ■I  a  solution  <>f  potassium  ferrocyanide  on  a  porcelain  tesjt- 
tablet  produces  instantaneously  a  brownish-red  coloration.1 
Take  the  burette  reading  and  calculate  the  P..O-  content  of  the 
urine  under  examination. 

Calculation. — Multiply  the  number  of  cubic  centimeter^  <<\ 
uranium  acetate  solution  used  by  0.005  t0  determine  the  num- 
ber of  grams  of  P2Ob  in  the  50  c.c.  of  urine  used.  To  express 
the  result  in  percentage  of  P2Or,  multiply  the  value  just  ob- 
tained by  -'.  e.  g.,  if  50  c.c.  of  urine  contained  0.074  gram  of 
PoOj  it  would  be  equivalent  to  0.148  per  cent. 

Calculate,  in  terms  of  P-O-j.  the  total  phosphate  content  of 
the  twenty-four  hour  urine  specimen. 

2.  Earthy  Phosphates. — To  100  c.c.  of  urine  in  a  beaker 

1  The  sodium  acetate  solution  is  prepared  by  dissolving  ioo  grams  of 
sodium  acetate  in  800  c.c.  of  distilled  water,  adding  100  c.c.  of  30  per  cent 
acetic  acid  to  the  solution  and  making  the  volume  of  the  mixture  up  to 
I  liter  with  water. 

1  This  uranium  acetate  solution  may  be  prepared  by  dissolving  35.461 
grams  of  uranium  acetate  in  one  liter  of  water.  One  c.c.  of  such  a  solu- 
tion should  be  equivalent  to  0.005  gram  of  P2O5,  phosphoric  anhydride. 
This  solution  may  be  standardized  as  follows :  To  50  c.c.  of  a  standard 
solution  of  disodium  hydrogen  phosphate,  of  such  a  strength  that  the  50 
c.c.  contains  0.1  gram  of  P-Os,  add  5  c.c.  of  the  sodium  acetate  solution, 
mentioned  above,  and  titrate  with  the  uranium  solution  to  the  correct 
end-reaction  as  indicated  in  the  method  proper.  Inasmuch  as  1  c.c.  of 
the  uranium  solution  should  precipitate  0.005  gram  of  P2O5,  exactly  20  c.c. 
of  the  uranium  solution  should  be  required  to  precipitate  50  c.c.  of  the 
standard  phosphate  solution.  If  the  two  solutions  do  not  bear  this  rela- 
tion to  each  other  they  may  be  brought  into  proper  relation  by  diluting 
the  uranium  solution  with  distilled  water  or  by  increasing  its  strength. 
A  10  per  cent  solution  of  potassium  ferrocyanide  is  satisfactory. 


368  PHYSIOLOGICAL    CHEMISTRY. 

add  an  excess  of  ammonium  hydroxide  and  allow  the  mixture 
to  stand  12-24  hours.  Under  these  conditions  the  phosphoric 
acid  in  combination  with  the  alkaline  earths,  calcium  and  mag 
nesium,  is  precipitated  as  phosphates  of  these  metals.  Col- 
.  lect  the  precipitate  on  a  filter  paper  and  wash  it  with  very 
dilute  ammonium  hydroxide.  Pierce  the  paper,  and  remove 
the  precipitate  by  means  of  hot  water.  Bring  the  phosphates 
into  solution  by  adding  a  small  amount  of  dilute  acetic  acid 
to  the  warm  solution.  Make  the  volume  up  to  50  c.c.  with 
water,  add  5  c.c.  of  sodium  acetate  solution  and  determine 
the  P205  content  of  the  mixture  according  to  the  directions 
given  under  the  previous  method. 

Calculation. — Multiply  the  number  of  cubic  centimeters  of 
uranium  acetate  solution  used  by  0.005  to  determine  the  num- 
ber of  grams  of  P205  in  the  100  c.c.  of  urine  used.  Since 
100  c.c.  of  urine  was  taken  this  value  also  expresses  the  per- 
centage of  P205  present. 

Calculate  the  quantity  of  earthy  phosphates,  in  terms  of 
P205,  present  in  the  twenty-four  hour  urine  specimen. 

The  quantity  of  phosphoric  acid  present  in  combination 
with -the  alkali  metals  may  be  determined  by  subtracting  the 
content  of  earthy  phosphates  from  the  total  phosphates. 

3.  Total  Phosphorus. — Sodium  Hydroxide  and  Potassium 
Nitrate  Fusion  Method. — Place  25  c.c.  of  urine  in  a  large 
silver  crucible  and  evaporate  to  a  syrup  on  a  water-bath.  Add 
10  grams  of  NaOH  and  2  grams  of  KN03  to  the  residue  and 
fuse  the  mass  until  all  organic  matter  has  disappeared  and  the 
fused  mixture  is  clear.  Cool  the  mixture,  transfer  it  to  a 
casserole  by  means  of  hot  water,  acidify  the  solution  slightly 
with  pure  nitric  acid  and  evaporate  to  dryness  on  a  water-bath. 
Moisten  the  residue  with  a  few  drops  of  dilute  nitric  acid,  dis- 
solve it  in  hot  water  and  transfer  to  a  beaker.  Now  add  an 
equal  volume  of  mplybdic  solution1  and  keep  the  mixture  at 
40°  C.  for  twenty-four  hours.  Filter  off  the  precipitate,  wash 
it  with  dilute  molybdic  solution  and  dissolve  it  in  dilute  am- 

1  Directions  for  the  preparation  of  the  solution  are  given  on  page  2>7- 


urine:  quantitative  analysis.  369 

monia.  Add  dilute  hydrochloric  acid  to  the  solution,  being 
careful  to  leave  the  solution  distinctly  ammoniacal.  Magn 
mixture2  (10  [5  c.c.)  should  now  be  added  and  after  stirring 
thoroughly  and  making  strongly  ammoniacal  with  concentrated 
ammonia  the  solution  should  l>e  allowed  to  stand  in  a  cool 
place  for  twenty-four  hours.  Filter  off  the  precipitate,  wash 
it  free  from  chlorine  by  means  of  dilute  ammonia  (1:5),  dry, 
incinerate  and  weigh,  as  magnesium  pyrophosphate,  MgJ'-Oj, 
in  the  usual  manner. 

In  thismethod  the  phosphoric  acid  of  the  urine  is  precipitated 
as  ammonium  magnesium  phosphate  and  in  the  process  of  in- 
cineration this  body  is  transformed  into  magnesium  pxrophos- 
phate. 

Calculation. — The  quantity  of  phosphorus,  expressed  in 
terms  of  P205,  in  the  volume  of  urine  taken  may  he  determined 
by  means  of  the  following  proportion  : 

Mol.  wt.  Wt  of  Mol.  wt 

Mg2P20, :  Mg2P207 : :  P206  :  x  (  wt.  of  P206  in  grams). 
ppt. 

If  31  represents  the  weight  of  the  Mg2P207  precipitate  and 
we  make  the  proper  substitutions  we  have  the  following  pro- 
portion : 

221.1:3?::  140.9:.!'  (  wt.  of  P205,  in  grams,  in  the  quantity  of 

urine  used). 

To  express  the  result  in  percentage  of  P205  simply  divide 
the  value  of  x,  as  just  determined,  by  the  quantity  of  urine  used. 

IX.    Creatinin. 

Folin's  Colorimetric  Method. — This  method  is  based  upon 

the  characteristic  property  possessed   alone  by  creatinin,   of 

yielding  a  certain   definite  color-reaction  in  the  presence  of 

picric  acid  in  alkaline  solution.     The  procedure  is  as  follows : 

2 Directions  for  the  preparation  of  magnesia  mixture  may  be  found  on 
page  2.-0. 
■     25 


37°  PHYSIOLOGICAL    CHEMISTRY. 

Place  10  c.c.  of  urine  in  a  500  c.c.  volumetric  flask,  add  15  c.c. 
of  a  saturated  solution  of  picric  acid  and  5  c.c.  of  a  10  per  cent 
solution  of  sodium  hydroxide  and  allow  the  mixture  to  stand 
for  5-6  minutes.  During  this  interval  pour  a  little  f  potas- 
sium bichromate  solution1  into  each  of  the  two  cylinders  of 
the  colorimeter  (Duboscq's)  and  carefully  adjust  the  depth  of 
the  solution  in  one  of  the  cylinders  to  the  8  mm.  mark.  A  few 
preliminary  colorimetric  readings  may  now  be  made  with  the 
solution  in  the  other  cylinder,  in  order  to  insure  greater  accu- 
racy in  the  subsequent  examination  of  the  solution  of  unknown 
strength.  Obviously  the  two  solutions  of  potassium  bichromate 
are  identical  in  color  and  in  their  examination  no  two  readings 
should  differ  more  than  0.1-0.2  mm.  from  the  true  value 
(8  mm.).  Four  or  more  readings  should  be  made  in  each  case 
and  an  average  taken  of  all  of  them  exclusive  of  the  first  read- 
ing, which  is  apt  to  be  less  accurate  than  the  succeeding  read- 
ings. In  time  as  one  becomes  proficient  in  the  technique  it  is 
perfectly  safe  to  take  the  average  of  the  first  two  readings. 

At  the  end  of  the  5-6  minute  interval  already  mentioned,  the 
contents  of  the  500  c.c.  flask  are  diluted  to  the  500  c.c.  mark, 
the  bichromate  solution  is  thoroughly  rinsed  out  of  one  of  the 
cylinders  with  the  solution  thus  prepared  and  a  number  of 
colorimetric  readings  are  immediately  made. 

Ordinarily  10  c.c.  of  urine  is  used  in  the  determination  by 
this  method  but  if  the  content  of  creatinin  is  above  15  mg.  or 
below  5  mg.  the  determination  should  be  repeated  with  a 
volume  of  urine  selected  according  to  the  content  of  creatinin. 
This  variation  in  the  volume  of  urine  according  to  the  content 
of  creatinin  is  quite  essential  since  the  method  loses  in  accuracy 
when  more  than  15  mg.  or  less  than  5  mg.  of  creatinin  is  pres- 
ent in  the  solution  of  unknown  strength. 

Calculation. — By  experiment  it  has  been  determined  that 
10  mg.  of  pure  creatinin,  when  brought  into  solution  and  di- 
luted to  500  c.c.  as  explained  in  the  above  method,  yields  a 
mixture  8.1    mm.  of  which  possesses  the  same  colorimetric 

JThis  solution  contains  24.55  grams  of  potassium  bichromate  to  the  liter. 


urine:  quantitative  analysis.  371 

value  as  8  mm.  of  a  g  solution  of  potassium  bichromate. 
Bearing  this  in  mind  the  computation  is  readily  made  by  means 
of  the  following  proportion  in  which  v  represents  the  number 
of  mm.  of  the  solution  of  unknown  strength  equivalent  to  the 
8  mm.  of  the  potassium  bichromate  solution: 

>y:S.i ::  10:*  (mgs.  of  creatinin  in  the  quantity  of  urine  used). 

This  proportion  may  be  used  for  the  calculation  no  matter 
what  volume"of  urine  (5,  10  or  15  c.c.)  is  used  in  the  deter- 
mination. The  10  represents  10  mg.  of  creatinin,  which  gives 
a  color  equal  to  8.1  mm.,  whether  dissolved  in  5.  10  or  15  c.c. 
of  fluid. 

Calculate  the  quantity  of  creatinin  in  the  twenty-four  hour 
urine  specimen. 

X.    Chlorides. 

1.  Mohr's  Method. — To  [OC.c.  of  urine  in  a  small  platinum 
or  porcelain  crucible  or  dish  add  about  2  grams  of  chlorine-free 
potassium  nitrate  and  evaporate  to  dryness  at  ioo°  C.  (The 
evaporation  may  be  conducted  over  a  low  flame  provided  care 
is  taken  to  prevent  loss  by  spurting-.)  By  means  of  crucible 
tongs  hold  the  crucible  or  dish  over  a  free  flame  until  all  car- 
bonaceous matter  has  disappeared  and  the  fused  mass  is  slightly 
yellow  in  color.  Cool  the  residue  somewhat  and  bring  it  into 
solution  in  a  small  amount  (15-25  c.c.)  of  distilled  water  acidi- 
fied with  about  10  drops  of  nitric  acid.  Transfer  the  solution 
to  a  small  beaker,  being  sure  to  rinse  out  the  crucible  or  dish 
very  carefully.  Test  the  reaction  of  the  fluid,  and  if  not 
already  acid  in  reaction,  to  litmus,  render  it  slightly  acid  with 
nitric  acid.  Now  neutralize  the  solution  by  the  addition  of 
calcium  carbonate  in  substance.1  add  2-5  drops  of  neutral 
potassium  chromate  solution  to  the  mixture  and  titrate  with  a 
standard  argentic  nitrate  solution.2 

1  The  cessation  of  effervescence  and  the  presence  of  some  undecomposed 
calcium  carbonate  at  the  bottom  of  the  vessel  are  the  indications  of  neutral- 
ization. 

3  Standard  argentic  nitrate  solution  may  be  prepared  by  dissolving 
29.060  grams  of  argentic  nitrate  in   1   liter  of  distilled  water.     Each  cubic 


37-  PHYSIOLOGICAL    CHEMISTRY. 

This  standard  solution  should  be  run  in  from  a  burette. 
stirring  the  liquid  in  the  beaker  after  each  addition.  The  end- 
reaction  is  reached  when  the  yellow  color  of  the  solution 
changes  to  a  slight  orange-red.  At  this  point  take  the  burette 
reading  and  compute  the  percentage  of  chlorine  and  sodium 
chloride  in  the  urine  examined. 

Calculation.- — Since  I  c.c.  of  the  standard  argentic  nitrate 
solution  is  equivalent  to  o.oio  gram  of  sodium  chloride,  to 
obtain  the  weight,  in  grams,  of  the  sodium  chloride  in  the  10 
c.c.  of  urine  used  multiply  the  number  of  cubic  centimeters  of 
standard  solution  used  by  o.oio.  If  it  is  desired  to  express  the 
result  in  percentage  of  sodium  chloride  move  the  decimal  point 
one  place  to  the  right. 

To  obtain  the  weight,  in  grams,  of  the  chlorine  in  the  10  c.c. 
of  urine  used  multiply  the  number  of  cubic  centimeters  of 
standard  solution  used  by  0.006,  and  if  it  is  desired  to  express 
the  result  in  percentage  of  chlorine  move  the  decimal  point  one 
place  to  the  right. 

Calculate  the  quantity  of  sodium  chloride  and  chlorine  in 
the  twenty-four  hour  urine  specimen. 

2.  Volhard-Arnold  Method. — Place  10  c.c.  of  urine  in  a 
100  c.c.  volumetric  flask,  add  20-30  drops  of  nitric  acid  (sp.gr. 
1.2)  and  2  c.c.  of  a  cold  saturated  solution  of  ferric  alum.  If 
necessary,  at  this  point  a  few  drops  of  an  8  per  cent  solution 
of  potassium  permanganate  may  be  added  to  dissipate  the  red 
color.  Now  slowly  run  in  the  standard  argentic  nitrate1  solu- 
tion (20  c.c.  is  ordinarily  used)  until  all  the  chlorine  has  been 
precipitated  and  an  excess  of  the  argentic  nitrate  solution  is 
present,  continually  shaking  the  mixture  during  the  addition 
of  the  standard  solution.  Allow  the  flask  to  stand  10  minutes, 
then  fill  it  to  the  100  c.c.  graduation  with  distilled  water  and 
thoroughly  mix  the  contents.  Now  filter  the  mixture  through 
a  dry  filter  paper,  collect  50  c.c.  of  the  filtrate  and  titrate  it  with 

centimeter  of  this  solution  is  equivalent  to  0.010  gram  of  sodium  chloride 
or  to  0.006  gram  of  chlorine. 

'  See  note   (2)   at  the  bottom  of  page  371. 


urine:  quantitative  analysis.  573 

standardized  ammonium  sulphocyanide  solution.1  The  first 
permanent  tinge  of  brown  indicates  the  end-point.  Take 
the  burette  reading  and  compute  the  weight  of  sodium  chlo- 
ride in  tin.'  [O  c.c.  1  if  urine  used. 

Calculation. — The  number  of  cubic  centimeters  of  am- 
monium sulphocyanide  solution  used  indicates  the  excess  of 
standard  argentic  nitrate  solution  in  the  50  c.c.  of  filtrate 
titrated.  Multiply  this  reading  by  _\  inasmuch  as  only  one- 
half  of  the  filtrate  was  employed,  and  subtracl  this  product 
from  the  number  of  cubic  centimeters  of  argentic  nitrate 
(  20  c.c. )  originally  used,  in  order  to  obtain  the  actual  number 
of  cubic  centimeters  of  argentic  nitrate  utilized  in  the  precipi- 
tation of  the  chlorides  in  the  10  c.c.  of  urine  employed. 

To  obtain  the  weight,  in  grams,  of  the  sodium  chloride  in  the 
10  c.c.  of  urine  used  multiply  the  number  of  cubic  centimeters 
of  the  standard  argentic  nitrate  solution,  actually  utilized  in 
the  precipitation,  by  0.010.  If  it  is  desired  to  express  the  re- 
sult in  percentage  of  sodium  chloride  move  the  decimal  point 
one  place  to  the  right. 

Jn  a  similar  manner  the  weight,  or  percentage  of  chlorine 
may  be  computed  using  the  factor  0.006  as  explained  in 
Mohr's  method,  page  371. 

Calculate  the  quantity  of  sodium  chloride  and  chlorine  in 
the  twenty-four  hour  urine  specimen. 

'This  solution  is  made  of  such  a  strength  that  i  c.c.  of  it  is  equal  to 
1  c.c.  of  the  standard  argentic  nitrate  solution  used.  To  prepare  the  solu- 
tion  dissolve  12.9  grams  of  ammonium  sulphocyanide,  NITSCN,  in  a 
little  less  than  a  liter  of  water.  In  a  small  flask  place  20  c.c.  of  the 
standard  argentic  nitrate  solution,  5  c.c.  of  the  ferric  alum  solution  and 
4  c.c.  of  nitric  acid  (sp.  gr.  1.2),  add  water  to  make  the  total  volume  100 
c.c.  and  thoroughly  mix  the  contents  of  the  flask.  Now  run  in  the  ammo- 
nium sulphocyanide  solution  from  a  burette  until  a  permanent  brown  tinge 
is  produced.  This  is  the  end-reaction  and  indicates  that  the  last  trace 
of  argentic  nitrate  has  been  precipitated.  Take  the  burette  reading  and 
calculate  the  amount  of  water  necessary  to  use  in  diluting  the  ammonium 
sulphocyanide  in  order  that  10  c.c.  of  this  solution  may  be  exactly  equal 
to  10  c.c.  of  the  argentic  nitrate  solution.  Make  this  dilution  and  titrate 
again  to  be  certain  that  the  solution  is  of  the  proper  strength. 


374  PHYSIOLOGICAL    CHEMISTRY. 

XI.     Acetone. 

Messinger-Huppert  Method. — Place  ioo  c.c.  of  urine  in 
a  distillation  flask  and  add  2  c.c.  of  50  per  cent  acetic  acid. 
Connect  the  flask  with  a  condenser,  properly  arrange  a  re- 
ceiver, attach  a  terminal  series  of  bulbs  containing-  water  and 
distil  over  about  nine-tenths  of  the  urine  mixture.  Remove  the 
receiver,  attach  another  and  subject  the  residual  portion  of  the 
mixture  to  a  second  distillation.  Test  this  fluid  for  acetone 
and  if  the  presence  of  acetone  is  indicated  add  about  100  c.c. 
of  water  to  the  residue  and  again  distil.  Treat  the  united 
acetone  distillates  with  1  c.c.  of  dilute  (12  per  cent)  sul- 
phuric acid  and  redistil,  collecting  this  second  distillate  in  a 
glass-stoppered  flask.  During  distillation,  however,  the  glass 
stopper  is  replaced  by  a  cork  with  a  double  perforation,  the 
glass  tube  from  one  perforation  passing  to  the  condenser, 
while  the  bulbs  containing'  water,  before-mentioned,  are  at- 
tached by  means  of  the  tube  in  the  other  perforation.  Allow 
the  distillation  process  to  proceed  until  practically  all  of  the 
fluid  has  passed  over,  then  remove  the  receiving  flask  and 
insert  the  glass  stopper.  Now  treat  the  distillate  carefully 
with  10  c.c.  of  a  yg-  solution  of  iodine  and  add  sodium  hydroxide 
solution,  drop  by  drop,  until  the  blue  color  is  dissipated  and 
the  iodoform  precipitates.  Stopper  the  flask  and  shake  it 
for  about  one  minute,  acidify  the  solution  with  concentrated 
hydrochloric  acid,  and  note  the  production  of  a  brown  color 
if  an  excess  of  iodine  is  present.  In  case  there  is  no  such 
excess,  the  solution  should  be  treated  with  -j-q  iodine  solution 
until  an  excess  is  obtained.  Retitrate  this  excess  of  iodine 
with  y$  sodium  thiosulphate  solution  until  a  light  yellow  color 
is  observed.  At  this  point  a  few  cubic  centimeters  of  starch 
paste  should  be  added  and  the  mixture  again  titrated  until 
no  blue  color  is  visible.     This  is  the  end-reaction. 

Calculation. — Subtract  the  number  of  cubic  centimeters 
of  yq  thiosulphate  solution  used  from  the  volume  of  j^  iodine 
solution  employed.  Since  1  c.c.  of  the  iodine  solution  is 
equivalent  to  0.967  milligrams  of  acetone,  and  since  1  c.c.  of 


urine:  quantitative  analysis.  375 

the  thiosulphate  solution  is  equivalent  to  1  c.c.  of  the  iodine 
solution,  if  we  multiply  the  remainder  from  the  above  sub- 
traction by  0.967  we  will  obtain  the  number  of  milligrams  of 
acetone  in  the  100  c.c.  of  urine  examined. 

Calculate  the  quantity  of  acetone  in  the  twenty-four  hour 
urine  specimen. 

XII.     /3-Oxybutyric  Acid. 

1.  Darmstadter's  Method. — This  method  is  based  on  the 
fact  that  crotonic  acid  is  formed  from  /3-oxybutyric  acid 
under  the  influence  of  concentrated  mineral  acids.  The 
method  is  as  follows :  Render  100  c.c.  of  urine  slightly  alka- 
line with  sodium  carbonate  and  evaporate  nearly  to  dryness 
on  a  water-bath.  Dissolve  the  residue  in  150-200  c.c.  of 
50—55  per  cent  sulphuric  acid,  transfer  the  acid  solution  to 
a  one  liter  distillation  flask  and  connect  it  with  a  condenser. 
Through  the  cork  of  the  flask  introduce  the  stem  of  a  drop- 
ping funnel  containing  water.  Heat  the  flask  gently  until 
foaming  ceases,  then  use  a  full  flame  and  distil  over  about 
300-350  c.c.  of  fluid,  keeping  the  volume  of  liquid  in  the  dis- 
tillation flask  constant  by  the  addition  of  water  from  the 
dropping  funnel  as  the  distillate  collects.  Ordinarily  it  will 
take  about  2-2^4  hours  to  collect  this  amount  of  distillate. 
Extract  the  distillate  two  or  three  times  with  ether  in  a  sepa- 
ratory  funnel,  evaporate  the  ether  and  heat  the  residue  at 
1600  C.  for  a  few  minutes  to  remove  volatile  fatty  acids. 
Dissolve  the  residue  in  50  c.c.  of  water,  filter  and  titrate  this 
aqueous  solution  of  crotonic  acid  with  T\  sodium  hydroxide 
solution,  using  phenolphthalein  as  indicator. 

Calculation. — One  c.c.  of  y0  sodium  hydroxide  solution 
equals  0.0086  gram  of  crotonic  acid,  1  part  of  crotonic  acid 
equals  1.2 1  part  of  /3-oxybutyric  acid,  and  1  c.c.  of  j6  sodium 
hydroxide  solution  equals  0.01041  gram  of  /3-oxybutyric  acid. 
To  compute  the  quantity  of  /?-oxybutyric  acid,  in  grams,  mul- 
tiply the  number  of  cubic  centimeters  of  fv  sodium  hydroxide 
solution  used  by  0.01041. 


376  PHYSIOLOGICAL    CHEMISTRY. 

2.  Bergen's  Method. — Render  100-300  c.c.  of  sugar-free1 
urine  slightly  alkaline  with  sodium  carbonate,  evaporate  the 
alkaline  urine  to  a  syrup  on  a  water-bath,  cool  the  syrup, 
rub  it  up  with  syrupy  phosphoric  acid  (being  careful  to  keep 
the  mixture  cool),  20-30  grams  of  finely  pulverized,  anhydrous 
cupric  sulphate  and  20-25  grams  of  fine  sand.  Mix  the  mass 
thoroughly,  place  it  in  a  paper  extraction  thimble2  and  extract 
the  dry  mixture  with  ether  in  a  Soxhlet  apparatus  (Fig.  125 
page  380).  Evaporate  the  ether,  dissolve  the  residue  in  about 
25  c.c.  of  water,  decolorize  the  fluid  with  animal  charcoal,  if 
necessary,  and  determine  the  content  of  /?-oxybutyric  acid  by 
a  polarization  test. 

3.  Boekelman  and  Bouma's  Method. — Place  25  c.c.  of 
urine  in  a  flask,  add  25  c.c.  of  12  per  cent  sodium  hydroxide 
and  25  c.c.  of  benzoyl  chloride,  stopper  the  flask  and  shake  it. 
vigorously  for  three  minutes  under  cold  zvater.  Remove  the 
clear  fluid  by  means  of  a  pipette,  filter  it  and  subject  it  to  a 
polarization  test.  Through  the  action  of  the  benzoyl  chloride 
all  the  lasvorotatory  substances  except  /3-oxybutyric  acid  will 
have  been  removed  and  the  lsevorotation  now  exhibited  by 
the  urine  will  be  due  entirely  to  that  acid. 

XIII.     Acidity. 

Folin's  Method. — The  total  acidity  of  urine  may  be  deter- 
mined as  follows :  Place  25  c.c.  of  urine  in  a  200  c.c.  Erlen- 
meyer  flask  and  add  15-20  grams  of  finely  pulverized  potas- 
sium oxalate  and  1-2  drops  of  a  1  per  cent  phenolphthalein 
solution  to  the  fluid.  Shake  the  mixture  vigorously  for  1-2 
minutes  and  titrate  it  immediately  with  ■%■$  sodium  hydroxide 
until  a  permanent  faint  pink  coloration  is  produced.  Take 
the  burette  reading  and  calculate  the  acidity  of  the  urine  under 
examination. 

Calculation. — If  y   represents  the  number  of  cubic  centi- 

1  If  sugar  is  present  it  must  be  removed  by  fermentation. 

2  The   Schleicher   and    Schiill   fat-free   extraction  thimble   is   very  satis- 
factory. 


urine:  quantitative  analysis.  377 

meters  of  ,N(I  sodium  hydroxide  used  and  y'  represents  the 
volume  of  urine  excreted  in  twenty  four  hours,  the  total 
acidity  of  the  twenty-four  hour  urine  specimen  may  be  cal- 
culated by  means  of  the  following  proportion: 

25  :  v  :  :  v':.r(  acidity  "I*  -\|  hour  urine  expressed  in  cubic  centi- 
meters of  TNU  sodium  hydroxide). 

Each  cubic  centimeter  of  ,\  sodium  hydroxide  contains 
0.004  gram  "of  sodium  hydroxide  and  this  is  equivalent  to 
O.O063  &ram  of  oxalic  acid.  'There fore,  in  order  to  express 
the  total  acidity  of  the  twenty- four  hour  urine  specimen  in 
equivalent  grams  of  sodium  hydroxide,  multiply  the  value  of 
x,  as  just  determined,  by  0.004,  or  multiply  the  value  of  x  by 
0.0063  if  it  is  desired  to  express  the  total  acidity  in  grams  of 
oxalic  acid. 

XIV.     Purin  Bases. 

Salkowski's  Method.— IMace  400-600  c.c.  of  proteid- 
free  urine  in  a  beaker.  Introduce  into  another  beaker  30-50 
c.c.  of  an  ammoniacal  silver  solution1  with  30-50  c.c.  of  mag- 
nesia mixture,2  add  some  ammonium  hydroxide  and  if  nec- 
essary some  ammonium  chloride  to  clear  the  solution.  Now 
add  this  solution  to  the  urine,  stirring  continually  with  a  glass 
rod,  and  allow  the  mixture  to  stand  for  one-half  hour.  Col- 
lect the  precipitate  on  a  filter  paper,  wash  it  with  dilute  am- 
monium hydroxide  and  finally  wash  it  back  into  the  original 
beaker.  Suspend  the  precipitate  in  600-800  c.c.  of  water,  add 
a  few  drops  of  hydrochloric  acid  and  decompose  it  by  means 
of  hydrogen  sulphide.  Now  heat  the  solution  to  boiling, 
filter  while  hot  and  evaporate  the  filtrate  to  dryness  on  a 
water-bath.  Extract  the  residue  with  20  30  c.c.  of  hot  3 
per  cent  sulphuric  acid  and  allow  the  extract  to  stand  twenty- 
four  hours.     Filter  off  the  uric  acid,  wash  it,  make  the  filtrate 

1  Prepared  by  dissolving  26  grams  of  silver  nitrate  in  about  500  c.c.  of 
water,  adding  enough  ammonium  hydroxide  to  redissolve  the  precipitate 
which  forms  upon  the  first  addition  of  the  ammonia  and  making  the 
balance  of  the  mixture  up  to  1  liter  with  water. 

2  Directions  for  preparation  may  be  found  on  page  -70. 


37$  PHYSIOLOGICAL    CHEMISTRY. 

ammoniacal  and  precipitate  the  purin  bases  again  with  silver 
nitrate.  Collect  this  precipitate  on  a  small-sized  chlorine-free 
filter  paper,  wash,  dry  and  incinerate  it  in  the  usual  manner. 
Now  dissolve  the  ash  in  nitric  acid  and  titrate  with  ammonium 
sulphocyanide  according  to  the  Volhard-Arnold  method  (seep. 
372).  Calculate  the  content  of  purin  bases  in  the  urine  exam- 
ined, bearing  in  mind  that  in  an  equal  mixture  of  the  silver 
salts  of  the  purin  bases,  such  as  we  have  here,  one  part  of  sil- 
ver corresponds  to  0.277  gram  of  nitrogen  or  to  0.7381  gram 
of  the  bases. 

XV.     Oxalic  Acid. 

Salkowski-Autenrieth  and  Barth  Method. — Place  the 
twenty- four  hour  urine  specimen  in  a  precipitating  jar,  add 
an  excess  of  calcium  chloride,  render  the  urine  strongly  am- 
moniacal, stir  it  well  and  allow  it  to  stand  18-20  hours. 
Filter  off  the  precipitate,  wash  it  with  a  small  amount  of 
water  and  dissolve  it  in  about  30  c.c.  of  a  hot  15  per  cent 
solution  of  hydrochloric  acid.  By  means  of  a  separatory 
funnel  extract  the  solution  with  150  c.c.  of  ether  which  con- 
tains 3  per  cent  of  alcohol,  repeating  the  extraction  four  or 
five  times  with  fresh  portions  of  ether.  Unite  the  ethereal 
extracts,  allow  them  to  stand  for  an  hour  in  a  flask  and  then 
filter  through  a  dry  filter  paper.  Add  5  c.c.  of  water  to  the 
filtrate,  to  prevent  the  formation  of  diethyl  oxalate  when  the 
solution  is  heated,  and  distil  off  the  ether.  If  necessary, 
decolorize  the  liquid  with  animal  charcoal  and  filter.  Con- 
centrate the  filtrate  to  3-5  c.c,  add  a  little  calcium  chloride 
solution,  make  it  ammoniacal  and  after  a  few  minutes  render 
it  slightly  acid  with  acetic  acid.  Allow  the  acidified  solution 
to  stand  several  hours,  collect  the  precipitate  of  calcium  oxa- 
late on  a  washed  filter  paper,1  wash,  incinerate  strongly  (to 
CaO)  and  weigh  in  the  usual  manner. 

Calculation. — Since  56  parts  of  CaO  are  equivalent  to  90 
parts  of  oxalic  acid,  the  quantity  of  oxalic  acid  in  the  volume 

1  Schleicher  and  Schiill,  No.  589,  is  satisfactory. 


urine:  quantitative  analysis.  379 

of  urine  taken  may  be  determined  by  multiplying  the  weight 

1  >f  ( '.i(  )  by  tin.'  factor  [.607 1 . 

XVI.     Total  Solids. 

1.  Drying  Method. —  Place  5  c.c.  of  urine  in  a  weighed 
shallow  dish,  acidify  very  slightly  with  acetic  acid  (1-3 
drops)  and  dry  it  in  vacuo  in  the  presence  of  sulphuric  acid, 
to  constant  weight.  Calculate  the  percentage  of  solids  in  the 
urine  sample  and  the  total  solids  for  the  twenty- four  hour 
period. 

Practically  all  the  methods  the  technique  of  which  includes 
evaporation  at  an  increased  temperature,  either  under  atmo- 
spheric conditions  or  in  vacuo  are  attended  with  error. 

2.  Calculation  by  Long's  Coefficient. — The  quantity  of 
solid  material  contained  in  the  urine  excreted  for  any  twenty- 
four  hour  period  may  be  approximately  computed  by  multi- 
plying the  second  and  third  decimal  figures  of  the  specific 
gravity  by  2.6.  This  gives  us  the  number  of  grams  of  solid 
matter  in  one  liter  of  urine.  From  this  value  the  total  solids 
for  the  twenty-four  hour  period  may  easily  be  determined. 

Calculation. — If  the  volume  of  urine  for  the  twenty-four 
hours  was  1120  c.c.  and  the  specific  gravity  1.018,  the  calcu- 
lation would  be  as  follows : 

(a)    18  X  2.6  =  46.8  grams  of  solid  matter  in  1  liter  of  urine. 

,,  N    46.8  X  1 120  ,.,  .  , 

{0)    -  =52.4  grams  solid  matter  111  1120  c.c.  of 

urine. 

Long's  coefficient  was  determined  for  urine  whose  specific 
gravity  was  taken  at  25 °  C.  and  is  probably  more  accurate, 
for  conditions  obtaining  in  America,  than  the  older  coefficient 
of  Haeser,  2.33. 


CHAPTER    XXII 


QUANTITATIVE  ANALYSIS  OF  MILK,  GASTRIC 
JUICE  AND  BLOOD. 

(a)    Quantitative  Analysis  of  Milk. 
I.  Specific    Gravity. — This    may    be    determined    conve- 
niently by  means  of  a  Soxhlet,  Veith  or  Quevenne  lactometer. 
A  lactometer  reading  of   32 °    denotes  a  specific  gravity  of 
Fig.  125.  1-032.       The    determination 

should  be  made  at  about  60  °  F. 
and  the  lactometer  reading  cor- 
rected by  adding  or  subtracting 
o.i°  for  every  degree  F.  above 
or  below  that  temperature. 

2.  Fat. —  (a)  Adams'  Paper 
Coil  Method. — Introduce  about 
5  c.c.  of  milk  into  a  small  beaker, 
quickly  ascertain  the  weight 
to  centigrams,  stand  a  fat-free 
coil1  in  the  beaker  and  incline 
the  vessel  and  rotate  the  coil  in 
order  to  hasten  the  absorption 
of  the  milk.  Immediately  upon 
the  complete  absorption  of 
the  milk  remove  the  coil  and 
again  quickly  ascertain  the 
weight  of  the  beaker.  The  dif- 
ference in  the  weights  of  the 
beaker  at  the  two  weighings  rep- 
resents the  quantity  of  milk 
absorbed  by  the  coil.  Dry  the 
coil  carefully  at  a  temperature 
below  ioo°  C.  and  extract  it 
with  ether  for  3-5  hours  in  a 

'Very  satisfactory  coils  are  manufactured  by  Schleicher  and  Schull. 

380 


SOX II LET    Al'I'AKAT US. 


QUANTITATIVE    ANALYSIS   OF    MILK. 


38l 


Im<;    126. 


N  -  - . 

tOl_  ! 

c 

»— 1 

— ' 


Soxhlet  apparatus  (Fig.  [25,  p.  380),  using  a  safety  water-bath, 
Heat  the  flask  containing  the  fat  to  constant  weight  at  a 
temperature  bel<  m  roo   C. 

Calculation. — Divide  the  weight  of   fat,  in  grams,  by  the 

:u  of  milk,  in  grams.     The  quotient  is  the  percentage  of 
fat  contained  in  the  milk  examined. 

>  Approximate  Determination  by  Feser's  LactOSCOpe. — 
Milk  is  opaque  mainly  because  of  the  suspended  fat  globules 
and  therefore"by  means  of  the  estimation  of  this 
opacity  we  may  obtain  data  as  to  the  approximate 
content  of  fat.  Feser's  lactoscope  (Fig.  126,  p. 
38]  1  may  be  used  for  this  purpose.  Pr 
as  follows:  By  means  of  the  graduated  pipette 
accompanying  the  instrument  introduce  4  c.c. 
l>\  milk  into  the  lactoscope.  Add  water  grad- 
ually, shaking  after  each  addition,  and  note  the 
point  at  which  the  black  lines  upon  the  inner 
white  glass  cylinder  are  distinctly  visible.  Ob- 
serve the  point  on  the  graduated  scale  of  the 
lactoscope  which  is  level  with  the  surface  of  the 
diluted  milk.  This  reading-  represents  the  per- 
centage of  fat  present  in  the  undiluted  milk. 
Pure  milk  should  contain  at  least  3  per  cent 
of  fat. 

3.  Total  Solids.1 — Introduce  2-5  grams  of  milk  into  a 
weighed  flat-bottomed  platinum  dish  and  quickly  ascertain  the 
weight  to  milligrams.  Expel  the  major  portion  of  the  water 
by  heating  the  open  dish  on  a  water-bath  and  continue  the 
heating  in  an  air-bath  or  water  oven  at  97 : -100  C.  until  the 
weight  is  constant.  (  This  residue  may  be  used  in  the  deter- 
mination of  ash  according  to  the  method  described  on  p.  382.  1 

1  The  percentage  of  total  solids  may  be  calculated  from  the  specific  gravity 
and  percentage  of  fat  by  means  of  the  following  formula  which  has  been 
proposed  by  Richmond : 

S  =  o.^5   L+1.2   F  +  0.14 
S  =  total  solids. 
L  =  lactometer  reading. 
F=  fat  content. 


J 


Feser's 
Lactoscope. 


382  PHYSIOLOGICAL    CHEMISTRY. 

Calculation. — Divide  the  weight  of  the  residue,  in  grams, 
by  the  weight  of  milk  used,  in  grams.  The  quotient  is  the  per- 
centage of  solids  contained  in  the  milk  examined. 

4.  Ash. — Heat  the  dry  solids  from  2-5  grams  of  milk, 
obtained  according  to  the  method  just  given,  over  a  very  low 
flame1  until  a  white  or  light  gray  ash  is  obtained.  Cool  the  dish 
in  a  desiccator  and  weigh.  (This  ash  may  be  used  in  testing 
for  preservatives  according  to  directions  on  page  195.) 

5.  Proteids. — Introduce  a  known  weight  of  milk  (5-10 
grams)  into  a  200-300  c.c.  Kjeldahl  digestion  flask  and  add 
20  c.c.  of  concentrated  sulphuric  acid  and  about  0.2  gram  of 
cupric  sulphate.  Expel  the  major  portion  of  the  water  by 
heating  over  a  low  flame  and  finally  use  a  full  flame  and 
allow  the  mixture  to  boil  1-2  hours.  Complete  the  deter- 
mination according  to  the  directions  given  under  Kjeldahl 
Method,  page  359. 

Calculation. — Multiply  the  total  nitrogen  content  by  the 
factor  6.37s  to  obtain  the  proteid  content  of  the  milk  ex- 
amined. 

6.  Casein. — Mix  about  20  grams  of  milk  with  40  c.c.  of 
a  saturated  solution  of  magnesium  sulphate  and  add  the  salt 
in  substance  until  no  more  will  dissolve.  The  precipitate 
consists  of  casein  admixed  with  a  little  fat  and  lacto-globulin. 
Filter  off  the  precipitate,  wash  it  thoroughly  with  a  saturated 
solution  of  magnesium  sulphate,3  transfer  the  filter  paper  and 
precipitate  to  a  Kjeldahl  digestion  flask  and  determine  the 
nitrogen  content  according  to  the  directions  given  in  the  pre- 
vious experiment. 

Calculation. — Multiply  the  total  nitrogen  by  the  factor 
6.37  to  obtain  the  casein  content. 

1  Great  care  should  be  used  in  this  ignition,  the  dish  at  no  time  being 
heated  above  a  faint  redness,  as  chlorides  may  volatilize. 

2  The  usual  factor  employed  for  the  calculation  of  proteid  from  the 
nitrogen  content  is  6.25  and  is  based  on  the  assumption  that  proteids 
contain  on  the  average  16  per  cent  of  nitrogen.  This  special  factor  of 
6.37  is  used  here  to  calculate  the  proteid  content  from  the  total  nitrogen, 
since  the  principal  proteid  constituents  of  milk,  i.  e.}  casein  and  lactalbumin. 
contain  15.7  per  cent  of  nitrogen. 

3Preserve  the  filtrate  and  washings  for  the  determination  of  lactalbumin. 


QUANTITATIVE    ANALYSIS    OF    GASTRIC    JUICE. 

7.  Lactalbumin. — To  the  filtrate  and  washings  from  the  de- 
termination of  casein,  as  jusl  explained,  add  Almen's  reagent1 
until  no  more  precipitate  forms.  Filter  oft"  the  precipitate 
and  determine  the  nitrogen  content  according  to  the  direc- 
tions given  under  Proteids,  page  382. 

Calculation. — Multiply  the  total  nitrogen  by  the  factor 
6.37  to  obtain  the  lactalbumin  content. 

8.  Lactose. — To  about  350  c.c.  of  water  in  a  beaker  add 
20  grams  of  milk,  mix  thoroughly,  acidify  the  fluid  with  about 
2  c.c.  of  10  per  cent  acetic  acid  and  stir  the  acidified  mixture 
continuously  until  a  flocculent  precipitate  forms.  At  this 
point  the  reaction  should  be  distinctly  acid  to  litmus.  Heat 
the  solution  to  boiling  for  one-half  hour,  filter,  rinse  the 
beaker  thoroughly  and  wash  the  precipitated  proteids  and 
the  adherent  fat  with  hot  water.  Combine  the  filtrate  and 
wash  water  and  concentrate  the  mixture  to  about  150  c.c. 
Cool  the  solution  and  dilute  it  to  200  c.c.  in  a  volumetric  flask. 
Titrate  this  sugar  solution  according  to  directions  given 
under  Fehling's  Method,  page  345. 

Calculation. — Make  the  calculation  according  to  directions 
p-iven  under  Fehling's  Method,  p.  34s,  bearing  in  mind  that  10 
c.c.  of  Fehling's  solution  is  completely  reduced  by  0.0676 
gram  of  lactose. 

(b)    Quantitative  Analysis  of  Gastric  Juice. 
Topfer's  Method. 

This  method  is  much  less  elaborate  than  many  others  but 
is  sufficiently  accurate  for  ordinary  clinical  purposes.  The 
method  embraces  the  volumetric  determination  of  (1)  total 
acidity,  (2)  combined  acidity,  and  (3)  free  acidity,  and  the 
subsequent  calculation  of  (4)  acidity  due  to  organic  acids 
and  acid  salts,  from  the  data  thus  obtained. 

Strain  the  gastric  contents  and  introduce  10  c.c.  of  the 
strained  material  into  each  of  three  small  beakers  or  porcelain 

1  Almen's  reagent  may  be  prepared  by  dissolving  5  grams  of  tannin  in 
240  c.c.  of  50  per  cent  alcohol  and  adding  10  c.c.  of  25  per  cent  acetic  acid. 


384  PHYSIOLOGICAL    CHEMISTRY. 

dishes.1  Label  the  vessels  A,  B  and  C,  respectively,  and  pro- 
ceed with  the  analysis  according  to  the  directions  given  below. 

1.  Total  Acidity.2 — Add  3  drops  of  a  1  per  cent  alcoholic 
solution  of  phenolphthalein3  to  the  contents  of  vessel  A  and 
titrate  with  j%  sodium  hydroxide  solution  until  a  dark  pink 
color  is  produced  which  cannot  be  deepened  by  further  addi- 
tion of  a  drop  of  y^"  sodium  hydroxide.  Take  the  burette 
reading  and  calculate  the  total  acidity. 

Calculation. — The  total  acidity  may  be  expressed  in  the 
following  ways : 

1.  The  number  of  cubic  centimeters  of  ^  sodium  hydrox- 
ide solution  necessary  to  neutralize  100  c.c.  of  gastric  juice. 

2.  The  weight  (in  grams)  of  sodium  hydroxide  necessary 
to  neutralize  100  c.c.  of  gastric  juice. 

3.  The  weight  (in  grams)  of  hydrochloric  acid  which  the 
total  acidity  of  100  c.c.  of  gastric  juice  represents,  i.  c,  per- 
centage of  HC1. 

The  forms  of  expression  most  frequently  employed  are  1 
and  3,  preference  being  given  to  the  former. 

In  making  the  calculation  note  the  number  of  cubic  centi- 
meters required  to  neutralize  10  c.c.  of  the  gastric  juice  and 
multiply  it  by  10  to  obtain  the  number  of  cubic  centimeters 
necessary  to  neutralize  100  c.c.  of  the  fluid.  If  it  is  desired  to 
express  the  acidity  of  100  c.c.  of  gastric  juice  in  terms  of  hydro- 
chloric acid,  by  weight,  multiply  the  value  just  obtained  by 
0.00365.4 

2.  Combined  Acidity.5 — Add  3  drops  of  sodium  alizarin 
sulphonate  solution6  to  the  contents  of  vessel  B  and  titrate 
with  ys  sodium  hydroxide  solution  until  a  violet  color  is  pro- 
duced.    In  this  titration  the  red  color,  which  appears  after 

1  If  sufficient  gastric  juice  is  not  available  it  may  be  diluted  with  water 
or  a  smaller  amount,  e.  g.,  5  c.c,  taken  for  each  determination. 

2  This  includes  free  and  combined  acid  and  acid  salts. 

3  One  gram  of  phenolphthalein  dissolved  in  100  c.c.  of  95  per  cent  alcohol. 
*  One  c.c.  of  yV  hydrochloric  acid  contains  0.00365  gram  of  hydrochloric 

acid. 
5 Hydrochloric  acid  combined  with  proteid  material. 
6  One  gram  of  sodium  alizarin  sulphonate  dissolved  in  100  c.c.  of  water. 


Ql   AMITATIVF.    ANALYSIS    OF    GASTRK     JUICE.  385 

the  tinge  of  yellow  duo  to  the  addition  of  the  indicator  has 
disappeared,  musl   be  entirely  replaced  by  a  distinct  violet 
color.     Take  the  burette  reading  and  calculate  the  combined 
acidity. 
Calculation. — Since  the  indicator  used  reacts  to  all  acidities 

except  combined  acidity,  in  order  to  determine  the  number 
of  cubic  centimeters  of  -j*  sodium  hydroxide  necessary  to 
neutralize  the  combined  acidity  of  10  c.c.  of  the  gastric  juice, 
we  must  subtract  the  burette  reading  just  obtained  from  the 
burette  reading  obtained  in  the  determination  of  the  total 
acidity.  The  data  for  100  c.c.  of  gastric  juice  may  be  calcu- 
lated according"  to  the  directions  given  under  Total  Acidity, 
page  384. 

3.  Free  Acidity.1 — Add  4  drops  of  di-methyl-amino-azo- 
benzene  (Topfer's  reagent)  solution2  to  the  contents  of  the  ves- 
sel C  and  titrate  with  y  0  sodium  hydroxide  solution  until  the 
initial  red  color  is  replaced  by  lemon  yellow.9  Take  the 
burette  reading  and  calculate  the  free  acidity. 

Calculation. — The  indicator  used  reacts  only  to  free  acid- 
ity, hence  the  number  of  cubic  centimeters  of  tNq  sodium 
hydroxide  used  indicates  the  volume  necessary  to  neutralize 
the  free  acidity  of  10  c.c.  of  gastric  juice.  To  determine  the 
data  for  100  c.c.  of  gastric  juice  proceed  according  to  the 
directions  given  under  Total  Acidity,  page  384. 

4.  Acidity  due  to  Organic  Acids  and  Acid  Salts. — This 
value  may  be  conveniently  calculated  by  subtracting  the  num- 
ber of  cubic  centimeters  of  y5  sodium  hydroxide  used  in  neu- 
tralizing the  contents  of  vessel  C  from  the  number  of  cubic 
centimeters  of  T^  sodium  hydroxide  solution  used  in  neutral- 
izing the  contents  of  vessel  B.  The  remainder  indicates  the 
number  of  cubic  centimeters  of  -£$  sodium  hydroxide  solution 
necessary  to  neutralize  the  acidity  due  to  organic  acids  and 
acid  salts  present  in  10  c.c.  of  gastric  juice.     The  data  for 

'Hydrochloric  acid  not  combined  with  proteid  material. 
2  One-half  gram  dissolved  in  100  c.c.  of  95  per  cent  alcohol. 
1  If  the  lemon  yellow  color  appears  as  soon  as  the  indicator  is  added  it 
denotes  the  absence  of  free  acid. 
26 


386  PHYSIOLOGICAL    CHEMISTRY. 

100  c.c.  of  gastric  juice  may  be  calculated  according  to  direc- 
tions given  under  Total  Acidity,  page  384. 

(c)    Quantitative  Analysis  of  Blood. 

For  the  methods  involved  in  the  quantitative  examination 
of  blood  see  Chapter  XL 


APPENDIX. 

Almen's  Reagent.1  —  Dissolve  5  grams  of  tannin  in  240  c.c. 
of  50  per  cent  alcohol  and  add  i«»  c.c.  of  -'5  per  cent  acetic 
acid. 

Ammoniacal  Silver  Solution.- — Dissolve  26  grams  of 
silver  nitrate  in  about  500  c.c.  of  water,  add  enough  ammo- 
nium hydroxide  to  redissolve  the  precipitate  which  forms  upon 
the  first  addition  of  the  ammonium  hydroxide  and  make  the 
volume  of  the  mixture  up  to  1  liter  with  water. 

Arnold-Lipliawsky  Reagent.'' — This  reagent  consists  of 
two  definite  solutions  which  are  ordinarily  preserved  sepa- 
rately and  mixed  just  before  using.  The  two  solutions  are 
prepared  as  follows  : 

1  (/ )    One  per  cent   aqueous   solution   of   potassium   nitrite. 

</>)  One  gram  of  ^-amino-acetophenon  dissolved  in  100 
c.c.  of  distilled  water  and  enough  hydrochloric  acid  (about  2 
c.c.)  added,  drop  by  drop,  to  cause  the  solution,  which  is  at 
first  yellow,  to  become  entirely  colorless.  An  excess  of  acid 
must  be  avoided. 

Barfoed's  Solution.4 — Dissolve  4  grams  of  cupric  acetate 
in  100  c.c.  of  water  and  acidify  with  acetic  acid. 

Baryta  Mixture.' — A  mixture  consisting  of  one  volume 
of  a  saturated  solution  of  barium  nitrate  and  two  volumes  of 
a  saturated  solution  of  barium  hydroxide. 

Boas'  Reagent." — Dissolve  5  grams  of  resorcin  and  3 
grams  of  saccharose  in  100  c.c.  of  95  per  cent  alcohol. 

Congo  Red." — Dissolve  0.5  gram  of  congo  red  in  90  c.c. 
of  water  and  add  10  c.c.  of  95  per  cent  alcohol. 

1  Ott'>  precipitation   test,  p.  297.     Determination  of  lactalbumin,  p.  383. 

2  Salkowski's  method,  page  377. 
'Arnold-Lipliawsky   reaction,  page  308. 
*  Barfoed's  test,  page   11. 

'Isolation  of  urea  from  urine,  page  -'4-'. 
'Test   fur   free  acid,  page  88. 
'Test   for  free  acid,  page  88. 

3*7 


388  PHYSIOLOGICAL    CHEMISTRY. 

Ehrlich's  Diazo  Reagent.1 — Two  separate  solutions  should 
be  prepared  and  mixed  in  definite  proportions  when  needed 
for  use. 

(a)  Five  grams  of  sodium  nitrite  dissolved  in  1  liter  of 
distilled  water. 

(b)  Five  grams  of  sulphanilic  acid  and  50  c.c.  of  hydro- 
chloric acid  in  1  liter  of  distilled  water. 

Solutions  a  and  b  should  be  preserved  in  well  stoppered  ves- 
sels and  mixed  in  the  proportion  1  :  50  when  required.  Green 
asserts  that  greater  delicacy  is  secured  by  mixing  the  solution 
in  the  proportion  1  :  100.  The  sodium  nitrite  deteriorates 
upon  standing  and  becomes  unfit  for  use  in  the  course  of  a 
few  weeks. 

Esbach's  Reagent.2 — Dissolve  10  grams  of  picric  acid 
and  20  grams  of  citric  acid  in  1  liter  of  water. 

Fehling's  Solution.3 — Fehling's  solution  is  composed  of 
two  definite  solutions — a  cupric  sulphate  solution  and  an 
alkaline  tartrate  solution,  which  may  be  prepared  as  follows : 

Cupric  sulphate  solution  =  34.64  grams  of  cupric  sulphate 
dissolved  in  water  and  made  up  to  500  c.c. 

Alkaline  tartrate  solution=  125  grams  of  potassium  hy- 
droxide and  173  grams  of  Rochelle  salt  dissolved  in  water  and 
made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber- 
stoppered  bottles  and  mixed  in  equal  volumes  when  needed 
for  use.     This  is  done  to  prevent  deterioration. 

Ferric  Alum  Solution.4 — A  cold  saturated  solution. 

Folin-Shaffer  Reagent.5 — This  reagent  consists  of  500 
grams  of  ammonium  sulphate,  5  grams  of  uranium  acetate 
and  60  c.c.  of  10  per  cent  acetic  acid  in  650  c.c.  of  distilled 
water. 

Furfurol  Solution.0 — Add  1  c.c.  of  furfurol  to  1000  c.c. 
of  distilled  water. 

1  Ehrlich's  diazo  reaction,  page  316. 

2  Esbach's  method,  page  344. 

3  Fehling's  method,  page  345.     Fehling's  test,  pages  8  and  286. 

4  Volhard-Arnold  method,  page  372. 
0  Folin-Shaffer  method,  page  349. 

8  Mylius's    modification    of    Pettenkofer's    test,    pages    122    and    301.      v. 
Udransky's  test,  pages  123  and  302. 


APPENDIX.  389 

Gallic  Acid  Solution.' — A  saturated  alcoholic  solution. 

Guaiac  Solution."' — Dissolve  0.5  gram  of  guaiac  resin  in 
30  c.c.  1  »f  95  per  cent  alo  >h<  »1. 

Giinzberg's  Reagent.3  — Dissolve  2  -ranis  of  phloroglucin 
and  1  gram  of  vanillin  in  ioo  c.c.  of  95  per  cent  alcohol. 

Hammarsten's  Reagent.' — Mix  1  voluble  of  25  per  cent 
nitric  acid  and  [9  volumes  of  25  per  cent  hydrochloric  acid 
and  add  1  vplume  of  this  acid  mixture  to  4  volumes  of  95 
per  cent  alcohol.  It  is  preferable  that  the  acid  mixture  be 
prepared  in  advance  and  allowed  to  stand  until  yellow  in  color 
before  adding  it  t<>  the  alcohol. 

Hopkins-Cole  Reagent.' — To  one  liter  of  a  saturated 
solution  of  oxalic  acid  add  00  grams  of  sodium  amalgam  and 
allow  the  mixture  t<>  stand  until  the  evolution  of  gas  ceases. 
Filter  and  dilute  with  _'  3  volumes  of  water. 

Hypobromite  Solution.6 — The  ingredients  of  this  solu- 
tion should  be  prepared  in  the  form  of  tzco  separate  solutions 
which  may  he  united  as  needed. 

(a)  Dissolve  125  grams  of  sodium  bromide  in  water,  add 
125  grams  of  bromine  and  make  the  total  volume  of  the  solu- 
tion 1  liter. 

(b)  A  solution  of  sodium  hydroxide  having  a  specific 
gravity  of  1.25.  This  is  approximately  a  22.^  per  cent 
solution. 

Preserve  both  solutions  in  rubber-stoppered  bottles  and  when 
needed  for  use  mix  equal  volumes  of  solution  a,  solution  b, 
and  water. 

Iodine  Solution.' — Prepare  a  2  per  cent  solution  of  potas- 
sium iodide  and  add  sufficient  iodine  to  color  it  a  deep  yellow. 

Jolles'  Reagent."  — This  reagent  has  the  following  com- 
posite in  : 

1  Gallic  acid  tot.  page  195. 

:  Guaiac  test,  page*   163,   191   and  331. 

8  Test  for  free  acid,  page  88. 

*  Hammarsten's  reaction,  pages  ui   and  300. 

Hopkins-Cole  reaction,  page  45. 
"  Methods  for  determination  of  urea,  page  351. 
'  Iodine  test,  page  24. 
8  Jolles'  reaction,  pages  48  and  292. 


39°  PHYSIOLOGICAL    CHEMISTRY. 

Succinic  acid   40  grams. 

Mercuric  chloride    20  grams. 

Sodium  chloride  20  grams. 

Distilled  water    1000  grams. 

Lugol's  Solution.1 — Dissolve  5  grams  of  iodine  and  10 
grams  of  potassium  iodide  in  100  c.c.  of  distilled  water. 

Magnesia  Mixture.2— Dissolve  175  grams  of  magnesium 
sulphate  and  350  grams  of  ammonium  chloride  in  1400  c.c. 
of  distilled  water.  Add  700  grams'  of  concentrated  ammo- 
nium hydroxide,  mix  thoroughly  and  preserve  the  mixture 
in  a  glass-stoppered  bottle. 

Millon's  Reagent.3 — Digest  1  part  (by  weight)  of  mer- 
cury with  2  parts  (by  weight)  of  HNOs  (sp.  gr.  1.42)  and 
dilute  the  resulting  solution  with  2  volumes  of  water. 

Molybdic  Solution.4 — Molybdic  solution  is  prepared  as 
follows,  the  parts  being  by  weight: 

Molybdic  acid   1  part. 

Ammonium  hydroxide    (sp.   gr.   0.96)    4  parts. 

Nitric  acid   (sp.  gr.   1.2)    15  parts. 

Morner's  Reagent5 — Thoroughly  mix  1  volume  of  for- 
malin. 45  volumes  of  distilled  water  and  55  volumes  of  con- 
centrated sulphuric  acid. 

Neutral  Olive  Oil.6 — Shake  ordinary  olive  oil  with  a  10 
per  cent  solution  of  sodium  carbonate,  extract  the  mixture 
with  ether  and  remove  the  ether  by  evaporation.  The  residue 
is  neutral  olive  oil. 

Nylander's  Reagent7 — Digest  2  grams  of  bismuth  sub- 
nitrate  and  4  grams  of  Rochelle  salt  in  100  c.c.  of  a  10  per 

1  Gunning's  iodoform  test,  page  304. 

2  Sodium  hydroxide  and  potassium  nitrate  fusion  method  for  determi- 
nation of  total  phosphorus,  page  368. 

B  Millon's  reaction,  page  44. 

1  Sodium   hydroxide  and  potassium  nitrate  fusion  method   for  determi- 
nation of  total  phosphorus,  page  368. 
".Morner's  test,  page  82. 
0  Emulsification  of  fats,  page  101. 
7  Nylander's  test,  pages  9  and  288. 


APPENDIX.  391 

cent  solution  of  potassium  hydroxide.  The  reagent  should 
then  be  ci "  iled  and  filtered* 

Obermayer's  Reagent.1 — A<1<1  2  4  grams  of  ferric  chlo- 
ride i"  a  liter  of  hydrochloric  acid  (sp.  gr.  [.19). 

Oxalated  Plasma.- — Allow  arterial  blood  to  run  into  an 
equal  volume  of  0.2  per  cent  ammonium  oxalate  solution. 

Paraphenelenediamine  Hydrochloride  Solution.'' — Two 
grams  dissolved  in  100  c.c.  of  water. 

Phenolphthalein/ — Dissolve  1  gram  of  phenolphthalein  in 
100  c.c.  of  95  per  cent  alcohol. 

Phenylhydrazin  Mixture.'' — This  mixture  is  prepared  by 
combining  1  part  of  phenylhydrazin-hydrochloride  and  2  parts 
of  -odium  acetate  by  weight.  These  are  thoroughly  mixed  in 
a  mortar. 

Phenylhydrazin-Acetate  Solution.'' — This  solution  is  pre- 
pared by  mixing  1  volume  of  glacial  acetic  acid,  1  volume  of 
water  and  2  volumes  of  phenylhydrazin  (the  base). 

Purdy's  Solution.7 — Purdy's  solution  has  the  following 
composition : 

Cupric   sulphate   4752  grams. 

Potassium   hydroxide    23.5       grams. 

Ammonia   (U.  S.  P..  sp.  gr.  0.9)    350.0      c.c. 

Glycerin    38.0       c.c. 

Distilled  water,  to  make  total  volume  1  liter. 

Roberts'  Reagent/ — Mix  i  volume  of  concentrated  nitric 
acid  and  5  volumes  of  a  saturated  solution  of  magnesium 
sulphate. 

Salted  Plasma.1' — Allow  arterial  blood  to  run  into  an  equal 
volume  of  a  saturated  solution  of  sodium  sulphate  or  a   10 

1  Obermayer's  test,  page  255. 

'  Experiments  on  blood  plasma,  page  167. 

3  Detection  of  hydrogen  peroxide,  page  196. 

4  Topfer's  method,  page  383. 

6  Phenylhydrazin    reaction,   pages   5   and   283. 
'*'  Phenylhydrazin   reaction,  pages  5   and   -'84. 
'  Purdy's  method,  page  347. 
v  Robert's   ring  test,  pages  48  and  291. 
'  Experiments  on  blood  plasma,  page  167. 


392  PHYSIOLOGICAL    CHEMISTRY. 

per  cent  solution  of  sodium  chloride.  Keep  the  mixture  in 
the  cold  room  for  about  24  hours. 

Schweitzer's  Reagent.1 — Add  potassium  hydroxide  to  a 
solution  of  cupric  sulphate  which  contains  some  ammonium 
chloride.  Filter  off  the  precipitate  of  cupric  hydroxide,  wash 
it  and  bring  it  into  solution  in  20  per  cent  ammonium  hy- 
droxide. 

Sherrington's  Solution.2 — This  solution  possesses  the  fol- 
lowing formula : 

Methylene-blue    0.1  gram. 

Sodium  chloride   1.2  gram. 

Neutral  potassium  oxalate    1.2  gram. 

Distilled  water  300.0  grams. 

Sodium  Acetate  Solution.3 — Dissolve  100  grams  of  so- 
dium acetate  in  800  c.c.  of  distilled  water,  add  100  c.c,  of  30 
per  cent  acetic  acid  to  the  solution  and  make  the  volume  of 
the  mixture  up  to  1  liter  with  distilled  water. 

Sodium  Alizarin  Sulphonate/ — Dissolve  1  gram  of  sodium 
alizarin  sulphonate  in  100  c.c.  of  water. 

Solera's  Test  Paper.5 — Saturate  a  good  quality  of  filter 
paper  with  0.5  per  cent  starch  paste  containing  a  little  iodic 
acid  and  allow  the  paper  to  dry  in  the  air.  Cut  it  in  strips  of 
suitable  size  and  preserve  for  use. 

Spiegler's  Reagent.6 — This  reagent  has  the  following  com- 
position : 

Tartaric   acid    20  grams. 

Mercuric  chloride   40  grams. 

Glycerin    100  grams. 

Distilled  water  1000  grams. 

Standard  Ammonium  Sulphocyanide  Solution.7 — This 
solution  is  made  of  such  a  strength  that  1  c.c.  of  it  is  equal 

1  Schweitzer's  solubility  test,  page  29. 

2  "Blood  counting,"  page  181. 

3  Uranium  acetate  method,  page  367. 

4  Topfer's  method,  page  383. 
0  Solera's  reaction,  page  38. 

"  Spiegler's  ring  test,  pages  48  and  291. 
7  Volhard-Arnold  method,  page  372. 


APPENDIX.  393 

to  i  c.c.  of  the  standard  argentic  nitrate  solution  mentioned 
below.  To  prepare  the  solution  dissolve  [2.9  grams  of  am- 
monium sulphocyanide,  NH4SCN,  in  a  little  less  than  a  liter 
of  water.  In  a  small  flask  place  20  C.C.  of  the  standard 
argentic  nitrate  solution.  5  c.c.  of  a  cold  saturated  solution 
of  ferric  alum  and  4  c.c.  of  nitric  acid  (sp.  gr.  i.j  ).  add 
water  to  make  the  total  volume  100  c.c.  and  thoroughly  mix 
the  content^  of  the  flask.  Now  run  in  the  ammonium  sulpho- 
cyanide solution  from  a  hurette  until  a  permanent  brown 
tinge  is  produced.  This  is  the  end-reaction  and  indicates 
that  the  last  trace  of  argentic  nitrate  has  been  precipitated. 
Take  the  burette  reading  and  calculate  the  amount  of  water 
necessary  to  use  in  diluting  the  ammonium  sulphocyanide  in 
order  that  10  c.c.  of  this  solution  may  be  exactly  equal  to  10 
c.c.  of  the  argentic  nitrate  solution.  Make  the  dilution  and 
titrate  again  to  be  certain  that  the  solution  is  of  the  proper 
strength. 

Standard  Argentic  Nitrate  Solution.1 — Dissolve  29.06 
grams  of  argentic  nitrate  in  1  liter  of  distilled  water.  Each 
cubic  centimeter  of  this  solution  is  equivalent  to  0.01  gram  of 
sodium  chloride  or  to  0.006  gram  of  chlorine. 

Standard  Uranium  Acetate  Solution." — Dissolve  35461 
grams  of  uranium  acetate  in  1  liter  of  water.  One  c.c.  of  such 
a  solution  should  be  equivalent  to  0.005  gram  of  P205,  phos- 
phoric anhydride. 

This  solution  may  be  standardized  as  follows :  To  50  c.c. 
of  a  standard  solution  of  disodium  hydrogen  phosphate,  of 
such  a  strength  that  the  50  c.c.  contains  0.1  gram  of  P205.  add 
5  c.c.  of  the  sodium  acetate  solution  mentioned  on  p.  392  and 
titrate  with  the  uranium  solution  to  the  correct  end-reaction 
as  indicated  in  the  method  proper  on  p.  367.  Inasmuch  as  1  c.c. 
of  the  uranium  solution  should  precipitate  0.005  gram  of  P205, 
exactly  20  c.c.  of  the  uranium  solution  should  be  required  to 
precipitate  the  50  c.c.  of  the  standard  phosphate  solution.     If 

1  Volhard-Arnold  method,  page  372.     Mohr's  method,  page  371. 

2  L'ranium  acetate  method,  page  367. 


394  rilYSIOLOGICAL    CHEMISTRY. 

the  two  solutions  do  not  bear  this  relation  to  each  other  they 
must  be  brought  into  the  proper  relation  by  diluting  the  ura- 
nium solution  with  distilled  water  or  by  increasing  its  strength. 

Starch  Iodide  Solution.1 — Mix  o.i  gram  of  starch  powder 
with  cold  water  in  a  mortar  and  pour  the  suspended  starch 
granules  into  75-100  c.c.  of  boiling  water,  stirring  continu- 
ously. Cool  the  starch  paste,  add  20-25  grams  of  potassium 
iodide  and  dilute  the  mixture  to  250  c.c.  This  solution  deteri- 
orates upon  standing,  and  therefore  must  be  freshly  prepared 
as  needed. 

Starch  Paste.2 — Grind  2  grams  of  starch  powder  in  a  mortar 
with  a  small  amount  of  water.  Bring  200  c.c.  of  water  to  the 
boiling-point  and  add  the  starch  mixture  from  the  mortar  with 
continuous  stirring.  Bring  again  to  the  boiling-point  and 
allow  it  to  cool.  This  makes  an  approximate  1  per  cent  starch 
paste  which  is  a  very  satisfactory  strength  for  general  use. 

Stokes'  Reagent.3 — A  solution  containing  2  per  cent  fer- 
rous sulphate  and  3  per  cent  tartaric  acid.  When  needed  for 
use  a  small  amount  should  be  placed  in  a  test-tube  and  am- 
monium hydroxide  added  until  the  precipitate  which  forms  on 
the  first  addition  of  the  hydroxide  has  entirely  dissolved.  This 
produces  ammonium  fcrrotartrate  which  is  a  reducing  agent. 

Tanret's  Reagent.4 — Dissolve  1.35  grams  of  mercuric 
chloride  in  25  c.c.  of  water,  add  to  this  solution  3.32  grams  of 
potassium  iodide  dissolved  in  25  c.c.  of  water,  then  make  the 
total  solution  up  to  60  c.c.  with  distilled  water  and  add  20  c.c. 
of  glacial  acetic  acid  to  the  mixture. 

Tincture  of  Iodine.5 — Dissolve  70  grams  of  iodine  and  50 
grams  of  potassium  iodide  in  1  liter  of  95  per  cent  alcohol. 

Toison's  Solution.6 — This  solution  has  the  following 
formula : 

1  Fehling's  method,  page  345. 

2  Experiments  on  starch,  page  24. 

3  Haemoglobin,  page  170.     Hsemochromogen,  page  173. 

4  Tanret's  test,  pages  48  and  293.' 
0  Smith's  test,  pages  122  and  301. 
""Blood  counting,"  page  181. 


APPENDIX.  395 

Methyl   violel   0.025  gram. 

Sodium  chloride   1.0      gram. 

Sodium   sulphate  8.0      grams 

Glycerin    30.0      grams. 

I  tistilled   water    [60.0 

Topfer's  Reagent.' — Dissolve  0.5  grams  of  di-methyl- 
amino-azobenzene  in   too  c.c.  of  95  per  cent  alcohol. 

Tropseolin  OO.-'  Dissolve  0.05  gram  of  tropaeolin  OO  in 
100  c.c.  of  go  per  cent  alcohol. 

Uffelmann's  Reagent." — Add  a  5  per  cent  solution  of  ferric 
chloride  to  a  1  per  cent  solution  of  carbolic  acid  until  an  ame- 
thyst-blue  col<  >r  is  obtained. 

1  Topfer's  methi  id,  page  383. 
1  Tesl  For  Free  acid,  page  89. 
'Uffelman's  reaction,  page  04. 


NDEX. 


Acetone,  28-',  302 

formula   for,  302 

Gunning's  iodoform  test  for,  304 

I  1  gal's  tesl   for,  305 

Lieben's  test  for,  305 

quantitative  determination  of,  374 

Reynolds-Gunning  test  for,  306 
Acholic  stool,    140 
AchrOO  dextrin,    22,    35 
Acid,  acetic,  238,  265 

alloxyproteic,  237,  259,  317 

amino-acetic,   71 

amino-ethyl-sulphonic,    118,    211 

a-amino-/3-hydroxy-propionic,    74 

a-amino-/3-imido-azol  -propionic, 
80 

a -ami  no-iso-butyl -acetic,  69 

a-amino-normal  glutaric,  70 

o-amino-propionic.  72 

amino-succinic,    70 

amino-valerianic,   73 

a-amino-iso-valerianic,    73 

aspartic,  65,   70,   107 

benzoic,  127,  237,  263 

butyric,  84,   194,  238,   265 

caproic,   187 

carbamic,    150 

cholic,   117 

chondroitin-sulphuric,     202,     237, 
259 

combined    hydrochloric,    84,    87 

cyanuric,  241 

a-e-di-amino-caproic,    78 

diazo-benzene-sulphonic,   317 

ethereal    sulphuric,    129,   237,   253 

fatty,  97,  99,   102 

formic.    238.    265 

free  hydrochloric,  84,  87 

glutamic.    65,    70,    107 

glycocholic,   117 

glycuronic,  16 

glycerophosphoric,    221,    238,    265 

glyoxylic,    45 

guaniain-o-amino-valerianic,    79 

hippuric.   127,  128,  237,  255 

homoeentisic.  9.  237,  262 

indol-amino-propionic,   77 

indoxyl-sulphuric,  237.   253 

inosinic,   207,   211 

kynurenic.  237,  262 


Acid,    lactic,    10.    84,    -'"7 
lauric.    187 
myristic.   187 
nucleic,  62 
oxalic,    237,    258 
oxaluric,    237,    264 
oxymandclic.  237,  262 
oxyproteic,  237,  259,  317 
palmitic,  97,  102,  103 
para  cresol-sulphuric,  237,  253 
para-oxyphenyl-acetic,     129,    136, 

237,  261 
para  oxyphenyl-a  amino-propionic, 

66 
para-oxyphenyl-propionic,         129, 

136.  237,  261 
paralactic,  208,  238,  265 
penaceturic,    238,    265 
phenol-sulphuric,    237,    253 
phenyl -a -amino-propionic.    73 
phosphocarnic.  207,  211,  238,  265 
pyrocatechin-sulphuric,    237,    253 
a-pyrrolidin-carboxylic.  74 
sarcolactic,  208 
skatol-carbonic,   135,   136 
skatoxyl-sulphuric,   237,  253 
sulphanilic,   316.    317 
tannic.   25,   28,  47 
taurocholic,  117 
uric.  9.  207,  237,  245 
uro ferric,   237,   259,   317 
uroleucic.  237,  262 
volatile   fatty,    129,    132,    194,   238 
Acid  albuminate,  55,  56 

coagulation  of,   55 
experiments   on,    56 
precipitation    of,    56 
preparation  of,  56 
solubility  of,   56 
sulphur  content  of,  56 
Acidity   of   gastric   juice,   quantitative 
determination  of,  383 
urine,  cause  of,  228 

quantitative        determination 
of.  376 
Acidosis,   cause   of,   309 
Acid-haematin,   173 

Acrolein,  formation  of,  from  olive  oil, 
100 
from  glycerin,  104 


397 


39« 


INDEX. 


Adams'   paper  coil  method   for   deter- 
mination of  fat  in  milk,  380 
Adamkiewicz  reaction,  45 
Adenin,  21 1,  238 
Adipocere,  99 
Adipolytic    enzymes,    34 
Alanin,   65,   72 
Albumin,  egg,  42,  51 

powdered,  preparation  of,  51 
tests   on,   51 

serum,  42,  52,  148,   149,  282, 

.  28q. 
Albumin   in  urine,   282,  289 

acetic     acid     and     potassium 

ferrocyanide   test   for,    293 

coagulation     or     boiling     test 

for,  292 
Heller's  ring  test   for,   290 
Jolles'   reaction   for,   292 
Robert's  ring  test  for,  291 
sodium    chloride    and    acetic 

acid  test  for,  293 
Spiegler's   ring   test   for,   291 
Tanret's   test   for,   293 
tests   for,   290 
Albuminate,   55 
acid,   55,    56 
alkali,   55,   57 
precipitation   of,   56 
sulphur  content  of,   56 
Albuminoids,  43,   62 
Albumoids,    43,   62 
Albumoses     (see     proteoses,     pp.     43, 

57.    59) 
Aldehyde,    1,    7 
Aldehyde  group,    18 
Aldehyde  test  for  alcohol,  21 
v.   Aldor's   method    of   detecting   pro- 
teose in  urine,  296 
Aldose,    1 

Alkali    albuminate,    55,    56,    57,    107 
experiments  on,   57 
precipitation  of,  57 
preparation    of,    57 
sulphur  content  of,  56 
Alkali-hrematin,    172 
Allantoin,   237,   259 

crystalline   form   of,  259 

experiments   on,    260 

formula    for,    259 

preparation    of,    from    uric    acid, 

260 
separation  of,  from  the  urine,  260 
Allen's  modification  of  Fehling's  test, 

287 
A  linen's   reagent,    preparation    of,    297 
Alloxyproteic  acid,   237,  259,   317 
Aloin-turpentine      test      for      "  occult 
blood,"    142,    144 


Amide,    definition    of,    65 
Amine,  definition  of,  65 
Amino  acids,  65,   129 
a-amino-/3-hydroxypropionic  acid,   74 
a-amino-/3-imido-azol-propionic      acid, 

80 
a-amino-iso-butyl-acetic  acid,   69 
a-amino-normal-glutaric  acid,  70 
Amino-succinic  acid,  70 
Amino-valerianic  acid,  7s 
a-amino-iso-valerianic   acid,    73 
Ammonia,   65,    107 
Ammonia  in  urine,  238,  270 

quantitative        determination 
of,  357 
Ammoniacal   silver  solution,  prepara- 
tion of,  377 
Ammoniacal-zinc     chloride     test     for 

urobilin,  268 
Ammonium       magnesium       phosphate 
("Triple  phosphate"), 
278 
in  urinary  sediments,  319 
Amphopeptone,  43 
Amyloid,  28,  29,  43,  62 
Amylolytic    enzymes,    34,     108 
Amylopsin,    108 

digestion   of   dry   starch   by,    109, 
114 
inulin  by,    114 
experiments  on,  112 
influence  of  bile  upon  action  of, 
114 
metallic     salts,    upon     action 
of,    113 
most    favorable    temperature    for 
action  of,  113 
Animal  parasites  in   feces,    143 

in    urinary    sediments,    339 
Anti-albumid,    86,    87 
Antipeptone,  43 
Appendix,  387 
Arabinose,   2,    16 

orcin  test  on,  17 
phenylhydrazin  test  on,   17 
Tollens'   reaction  on,    16 
Arginin,  65,  78,  107 
Arnold-Lipliawsky    reaction    for    dia- 
cetic  acid,   308 
reagent,    preparation    of,    308 
Aromatic  oxyacids,  237,  261 
Asparagin,  70 
Aspartic  acid,   65,   70 

crystalline  form  of,  70 
formula  for,  70 
Ash   of  milk,  quantitative  determina- 
tion of,  382 

Barfoed's  reagent,  preparation  of,   n 


[NDEX. 


Barfoed's  test   For  dextrose,   u,  289 

Baryta    mixture,    preparation    of,    242 
Bayberry     tallow,     saponification     of. 

102 
Beckmand-Heidenhain  apparatui 
"  Bence  Joins'  proteid,"  detection  of, 

296 
Benzoic  acid,  u;,  237 

crystalline    form    of,    264 

experiments   upon, 
formula    for,   263 
ilulity   of,  263 

sublimation   of,  263 

Berthelot-AtwatCr    bomb    calorimeter, 

366 
Bergell's    method     for    determination 

of  /9-oxybutyric  acid,  376 
Bile.  1 16.  282,  300 

constituents  of,    1  1  7 
daily  secretion  of,   116 
freezing-point  of.   11- 
influence  on  digestion,  gastric,  93 
pancreatic.    112,    114 
inorganic  constituents  of,  117,  121 
nucleo-proteid   of,    121 
reaction  of,   116,   121 
secretion  of.   1 16 
specific  gravity  of,  117 
Bile   acids     1  1  7 

Hay's   test    for,    123 
Mylius's  test  for,   122 
{Jeukomm's  test  for,  122 
Pettenkofer's  test  for,   122 
tests   for,    122 
v.  Udransky's  test  for,   123 
Bile  acids  in  feces,  detection  of,    146 
Bile  acids  in  urine,  2S2 

Hay's  test  for,  302 
Mylius's  test  for,  301 
Xeukomm's  test  for.  301 
Pettenkofer's     test     for, 

301 
Salkowski's  test  for,  302 
tests  for,  301 
v.    Udransky's    test    for, 
302 
Bile   pigments,    118 

Gmelin's  test  for,  121 
Hammarsten's    reaction    for, 

121 
Huppert's    reaction    for,    121 
Rosenbach's  test  for.    121 
Smith's  test  for,    122 
tests  for,  121 
Bile  pigments  in  urine,  282,  300 

Gmelin's  test  for,  300 
Hammarsten's      reaction 
for,  300 


Bile    pigments    in    urine,     rluppert'i 
reaction   for, 
Rosenba*  h'a  ti  -<  for,  300 

Smith's    test    for,    301 
300 

Bile  salts,  crystallization  of,   123 

Biliary  calculi.   120 

analysis    of,    124 

Bilicyanin,  118,  120 

Bilifuscin,    1  is 

Bilihumin,  118 

Biliprasin,    118 

Bilirubin,  118.  119 

crystalline  form  of,    1  tg 
in   urinary   sediments,   326 

Biliverdin,  1 1 8,  120 
"Biological"  blood  test,    159 
Biuret,  46.  241 

formation  of,  from  urea.  46.  241, 
243 
Biuret  test.  45 

Posner's   modification   of,   46 
Blood,  148,  282,  297 

Bordet  test  for,   159 

clinical   examination  of.    174 

coagulation  of.   157 

constituents  of,   148,  150 

defihrinated,   160 

detection    of,     158,     159,     163,     168 

erythrocytes  of,    150 

experiments  on,   160 

form    elements    of,    148 

guaiac  test  for,  158,  163 

haemin  test  for,  158,  163 

oxyhemoglobin   of,    155 

"occult,"  in   feces,    142,   144 

in  urine,  282,  297 

leucocytes  of,   156 

medico-legal  tests  for,  158 

microscopical       examination      of, 
160,    168 

nucleo-proteid   of,    148,    149 

pigment  of,   155 

plaques,  148 

plasma,  148,  149,  167 

preparation  of  hxmatin  from,  165 

preparation    of   laky,    161 

quantitative   analysis  of,   386 

reaction  of,   148,   160 

serum,   150,   166 

specific  gravity  of,  148,   160 

spectroscopic  examination  of,  169 

test  for  iron  in,   161 

total  amount  of,  148 

Zeynek  and  Xencki's  haemin  test 
for,    163 
Blood  casts  in  urine,  334 


400 


INDEX. 


Blood  corpuscles,   148,   150,   156,   168 

"  counting,"    180 
Blood   in   urine,   282,   297 

guaiac   test    for,    299 
Teichmann's  hremin  test  for, 

298 
Heller's  test  for,  298 
Heller-Teichmann       reaction 

for,   298 
Schalfijew's   hsemin    test    for, 

299 
spectroscopic         examination 

of,    299 
tests    for,    298 

Zeynek   and   Nencki's   hsemin 
test  for,  299 
Blood  plasma,  148,  149,  167 
constituents   of,   148 
crystallization    of    oxyhemo- 
globin of,   156,   167 
effect    of    calcium     on     oxa- 

lated,    167 
experiments  on,   167 
preparation       of       fibrinogen 
from,   167 

oxalated,    167 
salted,    167 
Blood   serum,    150,    166 

coagulation    temperature    of, 

166 
constituents  of,    150 
experiments  on,   166 
precipitation    of   proteids    of, 

166 
separation    of    albumin    and 

globulin    of,     166 
sodium  chloride  in,  166 
sugar  in,   166 
Blood  stains,  examination  of,  168 
Boas'    reagent,    as    indicator,    88 

preparation  of,  88 
Boekelman   and   Bouma's  method   for 
determination  of  /3-oxybutyric  acid, 
376 
Boettger's  test  for  sugar,  9,  288 
Bomb  calorimeter,  Berthelot-Atwater, 

366 
Bone,  constituents  of,  204 

ossein  of,  preparation  of,  204 
Bone    ash,    scheme    for    analysis    of, 

205 
Bordet  test,  detection  of  human  blood 

by,  159 
Boric   acid   and   borates   in   milk,   de- 
tection of,  196 
Buccal  glands,   32 
Butyric  acid,  84,   194 


Butyrin,  98,  187 

Cadaverin,   78 

Calcium     and     magnesium     in     urine, 
238,    279 
carbonate    in    urinary    sediments 

319,  321 
casein,  188 
oxalate,  319 

in   urinary   sediments,    320 
phosphate    in    urinary    sediments, 
321 
in   milk,    193 
sulphate     in     urinary     sediments, 
322 
Calculi,  biliary,    120 
urinary,  340 

calcium  carbonate  in,  341 

oxalate  in,  341 
cholesterin  in,   342 
cystin  in,   342 
fibrin  in,   342 
indigo   in,   342 
phosphates  in,   341 
uric   acid   and  urates   in,   341 
urostealiths    in,    342 
xanthin  in,   342 
Cane  sugar   (see  saccharose,  p.   19) 
Caproic  acid,   187 
Carbamic  acid,   150 
Carbohydrates,    1 

classification  of,    1 
composition   of,    1,   2 
review  of,  29 

scheme  for  detection   of,   31 
variation  in  solubility  of,  2 
Carbonates  in  urine,  238,  280 
Carbon   monoxide  haemoglobin,    171 
Carnin,  207 
Carnosin,  207,  211 
Cartilage,    202 

constituents  of,  202 
experiments   on,   203 
Hopkins-Cole  reaction   on,  203 
loosely  combined  sulphur  in,   203 
Millon's  reaction  on,   203 
preparation  of  gelatin  from,  203 
solubility  of,  203 
xanthoproteic  test  on,   203 
Casein,  188 

soluble,   188 
calcium,  188 

quantitative      determination      of, 
382 
Caseinogen,    146,    187,    190 

action  of  rennin  upon,  188 
biuret  test  on,  193 


INDEX. 


4OI 


aogen,    Millon'a  tesl   on,    193 
precipitation  oi 
preparation  of,  19a 
solubility  of,   193 
tesl  for  loosely  combined  sulphur 

in,    193 
test  for  phosphorus  in,  193 

.!-'x.    333 

blood,  334 
epithelial,  334 
fatty,  334 
granular,  333 

hyaline. 

pus,  336 
waxj 

Casts  in  urinary  sediments,  328,  332 
Cellulose,  2,  28 

action     of    Schweitzer's     reagent 

on,  29 
hydrolysis   of,    29 
iodine  test  on,  28 
solubility  of,  28 
Cellulose  group,  2 
Cerebrin,  220 

experiments   on,    224 

hydrolysis  of,  224 

microscopical       examination      of, 

224 
preparation  of,  224 
solubility  of,  224 
Charcot-Leyden  crystals,   142 

form   of,    141 
Chlorides   in   urine,  238,   274 
detection  of,  275 
quantitative        determination 
of,    371 
Cholecyanin,   120 

Cholera-red    reaction    for    indol.     137 
Cholesterin.   121,   124,  222 
crystalline   form  of,    125 
formula  for,  222 
iodine-sulphuric     acid     test     for, 

124,  223 
isolation   of,  from   biliary  calculi, 

124 
Liebermann-Burchard     test     for, 

124,  224 
occurrence    of,    in    urinary    sedi- 
ments. 319,  325 
preparation  of,  from  nervous  tis- 
sue, 223 
Salkowski's  test  for,   125,  224 
Schiffs   reaction   for,    125,   224 
tests  for,    124.  223 
Choletelin,    118 
Cholin,  221 
Chondrigen.   62 

27 


(  hondroalbumoid,  4.*.  202 

1  Ihondromucoid,  6a,  202 

( Ihondroitin,  202 

Chondroitia  sulphuric   acid,   202,  237, 

259 
Chondrosin,  202 
Cipollina's  test,  6,  284 

Coagulated   proteids,  60 

biuret   test    on,   6l 
formation    of,   60 
Hopkins-Cole  reaction  on,  61 
Millon's  reaction  on,   61 
solubility   of,   61 
xanthoproteic     reaction     on, 
61 
Coagulation  of  proteids,   49,   50,   60 

changes  in  composition  dur- 
ing, 60 
fractional,    60 
Coagulation   temperature   of   proteids, 
50 
apparatus   used   in   determin- 
ing,  50 
method    employed    in    deter- 
mining,   50 
Collagen,  43,  62,   198,   199 
experiments   on,    199 
hydrolysis  of,  200 
percentage   of,  in   ligament,   201 

in  tendon,   198 
production  of  gelatin  from,  200 
solubility  of,  199 
Colostrum,  190 

microscopical   appearance   of.    188 
Combined  hydrochloric  acid,  84,  87 

tests  for,  87-90 
Compound  proteids,  43,  61 
classes  of,  61 
experiments  on,  199 
nomenclature  of,  61 
occurrence  of,  62 
Compound   test   for   lactose   in   urine, 

3i3 
Congealing-point   of    fat,    105 
Congo  red,  as  indicator,  88 
preparation  of,  88 
Conjugate  glycuronates,  9,  282,   310 
fermentation-reduction      test 

for,  310 
ToIIens'    reaction    on,    310 
Connective   tissue,    197 
Creatin,   150 

crystalline  form  of,  209 
formula    for,    210 
separation  of,  from  meat  extract, 
215 
Creatinin,  q,  207.  237,  250 
crystalline   form  of,   251 


40: 


INDEX. 


Creatinin,   daily   excretion   of.   250 
experiments  on,  251 
formula  for.  210,  250 
Jaffe's  reaction  for,  253 
quantitative      determination      of, 

369 

Salkowski's  test  for,  253 

separation  of,  from  urine,  251 

Weyl's  test  for,  J52 
Creatinin-zinc  chloride,  formation  of, 

250,  252 
Cresol,  para,  65,  129,  132 

tests  for,   138 
Cryoscopy,   232 
Cul-de-sac,  84 
Cyanuric  acid,  241 

formula  for.  241 
Cylindroids  in  urinary  sediments,  337 
Cystin,  65.  76.  237,  324 

crystalline  form  of,   76,  325 

detection  of,  325 

formula  for,   76 

in  urinary  sediments,   324 

Dare's   hjemoglobinometer,    178 
description  of,  178 
determination        of       haemo- 
globin by.    179 
Darmstadter's  method  for  determina- 
tion of  /3-oxybutyric  acid,  375 
Decomposition    products    of   proteids, 
65 
crystalline  forms  of,  68-79 
experiments  on,  80 
isolation  of,  80 
Delusive   feeding    experiments,    83 
Detection    of    preservatives    in    milk, 
195 
boric  acid  and  borates,   196 
formaldehyde,   195 
hydrogen  peroxide,   196 
salicylic  acid  and  salicylates,   195 
Deuteroproteose,  43 
Dextrin,  2,  27 

achroo-,   22,    35 
erythro-.   22,   27,   35 
action   of  tannic  acid   on,   28 
diffusibility   of,   28 
Fehling's  test   on,   27 
hydrolysis  of,  27 
iodine  test  on,  27 
solubility  of,  27 
Dextrosazon,      crystalline      form      of, 

Plate  III,  opposite  p.  5. 
Dextrose,  1.  3.  4,  282 

Allen's   modification    of   Fehling's 

test  for,  287 
Barfoed's  test  for,  11,  289 
Boettger's  test  on,  9,  288 


Dextrose,    Cipollina's   test   on,    6,    284 
diffusibility  of,  6 
Fehling's  test  on,  8,  2S6 
fermentation  of.   10,  288 
iodine  test  on.  6 
Molisch's  reaction  on,   4 
Moore's  test  on,  7 
Xylander's  test  on,  9,  288 
phenylhydrazin   test   on,   5,   283 
quantitative  determination  of,  345 
reduction  tests  on,  7,  284 
solubility   of,    4 
Trommer's  test  on,  8,  285 
Diacetic   acid,   282,   306 

Arnold-Lipliawsky    test    for, 

308 
formula  for,   306 
Gerhardt's  test   for,   307 
a-e-di-amino-caproic  acid,  78 
Diastase,  18 
Diazo-benzene-sulphonic   acid.    317 

reagent,    preparation    of, 
316 
Diazo   reaction    (Ehrlich's).   316 
Differentiation     between     pepsin     and 

pepsinogen.  92 
Digestion,    gastric.   83 
pancreatic,   106 
salivary,    32 
Di-methyl-amino-azobenzene  (see  Top- 

fer's  reagent),  88 
Disaccharides,   17 

classification  of,  2 
Doremus-Hinds  ureometer,  355 
Drying   method   for   determination   of 
total  solids  in  urine,  379 

Earthy  phosphates  in  urine,  275 

quantitative        determination 
of,    367 
Edestin,  53,  54 

coagulation  of,  54 

crystalline   forms   of,    54 

microscopical   examination  of,   54 

Millon's  test  on,  54 

preparation  of,   53 

solubility  of,   54 

tests  on  crystallized.   54 
filtrate  of,  54 
Ehrlich's  diazo-benzene-sulphonic  acid 

reagent,  preparation  of,   316 
Ehrlich's  diazo  reaction,   316 
Einhorn's   saccharometer,    10 
Elastin,  43,   62,    198  201 

experiments  on,  201 

preparation    of.    201 

solubility   of,   201,   202 
Electrical  conductivity  of  urine,  235 
Enterokinase.    108 


403 


Enzymes,  3  \.  238 

classification  ol 

uanin,  238 
Episarkin, 
Epithelial  cells  in  urinary  sediments, 

casts  in   urinary   sediments,   328, 

33*i 
Epithelial  tissue,  107 

experiments  on,    1  •  j  r 
Erythrocytes,    [48,    150,  151 
counting   the,    180 
diameter  af,   151 
form  of,  1  so 
influence  of  osmotic  pressure  on, 

[62 
in  urinary  sediments,  .12%,  337 
number  of,  per  cubic  mm.,   151 
of  different  species,  1 5 1 
stroma  of,   151,  156 
variation  in  number  of,   151 
Erythro-dextrin,   22,   27.   34,   3s 
Esbach*s  albuminometer,  345 

method      for     determination     of 

albumin,   .144 
reagent,  preparation  of,   344 
Ester,    definition    of,   97 
hydrochloric  acid.    165 
sulphuric  acid,    165 
Ethereal  sulphates,  272,  273 

quantitative        determination 
of.    363 
Ethereal  sulphuric  acid.   129,  237,  253 
Euglobulin,    149 

Extractives  of  muscular  tissue,  207 
nitrogenous,  207 
non-nitrogenous.    207 

Fats,  96 

absorption  of,  99 

apparatus    for    determination     of 

melting-point    of,     103 
boiling-point  of,  98 
chemical    composition   of,    97 
congealing-point  of,  105 
crystallization  of,  98,   101 
digestion   of,  99 
emulsification   of,   99,    101 
experiments  on,    100 
formation   of  acrolein  from,  100 
hydrolysis    of,    97 
in  milk,   187,   194 
in  urine.   2S2,   312 
melting-point  of,  98,   104 
nomenclature   of.   98 
occurrence  of.  96,  98 
permanent   emulsions   of,   99,    101 
quantitative      determination      of, 

in    milk.    380 


Fats,  reaction  of,   i"" 

nification  of,  '17.  102, 

solubility  of,  98,   

transitory  emulsions  of,  'n,   i"i 
Fatty  acid.  97,  99,   1  oa 
Fatty     casts     in     urinary     sediments, 

328,    3.U 

Fatty  degeneration,  99 

1 39 

blood   in,    142 

daily  excretion  of,  139 
detection  of  albumin  and  globulin 
in,    147 
Kill-   acids   in,    146 
bilirubin   in,    145 
caseinogen   in,    146 
cholestcrin    in,    143 
hydrobilirubin  in,  145 
inorganic      constituents       of, 

'  17 
nucleo  pr'oteid   in.    146 
proteose  and  peptone  in,    147 
experiments  on.  142 
form   and  consistency  of,   141 
macroscopic  constituents  of.  141 
microscopic    constituents    of,     14! 
odor  of.    140 
pigment   of,    140 
reaction  of.    141 

plitting  enzymes,  86,  97,  99,  109 
Fehling's  method  for  determination  of 
dextrose.  345 
solution,  preparation  of,  8,  2S6 
test,  8,   286 

Allen's    modification    of.    287 
Ferments,  classification  of.   34 
Fermentation    test,    10,    288 
Fermentation    method    for   determina- 
tion of  dextrose,  3  (.8 
Fermentation-reduction   test   for  con- 
jugate   glycuronatcs.    310 
Ferric  chloride  test  for  sulphocyanide 

in  saliva.  37 
Fibrin,    1411,    168,   282 

in    urinary    sediments,    328,    339 
separation  of,  from  blood,  168 
solubility  of,  168 
Fibrin    ferment,    150.    158 
Fibrinogen,  1 4.8,  149,  158 
Fischer   apparatus,   photograph   of,   67 
1  'leischl's  haemometer,  1 74 
description   of.    174 
determination  of  haemoglobin 
by.   175 
Fleischl-Miescher  ha?mometer,    176 
Fluorides  in  urine.  23S.   280 
Fly-maggots,  experiments  on,  100 
Folin's    method    of    determination    of 
acidity   of   urine.    376 


404 


INDEX. 


Folin's    method    of    determination    of 
acidity     of     ammonia. 
357 
creatinin,   369 
ethereal  sulphates,  363 
inorganic    sulphates.    362 
total   sulphates,   361 
urea,   355 
Folin-Shaffer    method    for    determina- 
tion of  uric  acid,   349 
Foreign    substances    in    urinary    sedi- 
ment. 328,   339 
Form  elements  of  blood,   148 
Formic  acid,  238,  265 
Fractional  coagulation  of  proteids,  60 
Free  hydrochloric  acid,  84,  87 

tests  for,  87-90 
Freezing-point  of  bile,   117 
blood,   148 
milk,   187 

pancreatic  juice,  107 
urine,  232 
Fuchsin-frog   experiment,   213 
Fundus  glands,  83 

Furfurol  solution,   preparation  of,  122 
Fusion  mixture,  preparation  of,  52 

Galactose.  2,   15 

experiments  on,  15 
Gallic  acid  test  for  formaldehyde,  195 
Gastric   digestion,   83 

conditions  essential  for,  85,  91 
general    experiments    on,    91 
influence  of  bile  on,  93 
influence    of    different    tem- 
peratures  on,   91 
most  favorable  acidity  for,  91 
power  of  different  acids  in,  92 
products   of,   85,   87 
Gastric  fistula,  83 
Gastric  juice,  83-86 

acidity   of,   84,   85 
artificial,    preparation    of,    86 
composition  of,  84 
enzymes    of,    84 
quantitative   analysis   of,   383 
quantity  of.   83 
reaction  of,  84 
specific  gravity  of.  84 
lactic  acid  in,  tests  for,  94 
Gelatin,  43,  62,  68,  198,  200 
coagulation  of,  200 
experiments  on,  200 
formation   of,   200 
Hopkins-Cole  reaction   on,   200 
Millon's  reaction  on,   200 
precipitation  of.  by  alcohol,   201 
alkaloidal    reagents,    200 
metallic  salts,   200 


Gelatin,    precipitation    of,    by    mineral 
acids,   200 
preparation     of,     from     cartilage, 
203 
from    collagen,    200 
salting-out   of,   200 
solubility  of,  200 
Gerhardt's  test  for  diacetic  acid,  307 
Gerhardt's   test   for  urobilin.    268 
Gliadin,   68,   69,    71,    73,    74,    76 
Globulin,  43,   53 

experiments   on,    53 
preparation  of,    53 
serum,   43,    148,    149,   282 
in  urine,  282,  289.  293 
tests  for,  293 
Glucoproteid,  43,  61,  199 
experiments  on,  .199 
hydrolysis  of,   199 
Glucose  (see  Dextrose,  p.  3) 
Glutamic  acid,  65,   70 

formula  for,   70 
Glutenin,  70,  72 
Glycerin,  97,  99,   104 

borax  fusion  test  on,  104 
experiments  on,    104 
formula  for,  97,   100 
Glycerin     extract     of    pig's     stomach, 

preparation  of,  87 
Glycerophosphoric  acid,  221,  238,  266 
Glycocholic  acid,   117 
Glycocholic  acid  group,  117 
Glycocoll,  65,  71,  117 

formula  for,   71,   117 
preparation  of,    127 
Glycocoll     ester    hydrochloride,     crys- 
talline form  of,   72 
Glycogen,  2,  26,  207,  208 
experiments    on,    215 
hydrolysis  of,  215 
influence  of  saliva  on,  215 
iodine  test  on,  215 
preparation  of.  215 
Glycosuria,  alimentary,  3 
Glycuronates,  conjugate,  9,  282,  310 
Glycuronic  acid,    16 
Glyoxylic  acid,  45 

formula  for,  45 
Gmelin's   test   for  bile   pigments,    121, 
300 
Rosenbach's   modification   of, 
121,  300 
Granular  casts  in  urinary  sediments, 

328,  333 
Granulose,   22 
Green  stools,  cause  of,  140 
Guaiac    solution,    preparation    of,    163 
Guaiac  test  on  blood,  158,   163 
milk,  191 


IX'iKX. 


40  5 


Guaiac  tesl  on  pus,  3.*' 
Guanidin-a-amino-valerianic    acid,    79 
( rtianin,  -■  i . 
tliiius  and   vegetable   mucilage   group 

i>\   carbohydrati 
Gunning's  iodoform   test   i"r  acetone, 

Gunzberg's    reagent,   as    indicator,    88 
preparation  of,  88 

Hsematin,  156 

acid-.    173 
alkali-.    172 
preparation   of,    165 
reduced   .alkali-.    173 
Hsematoidin,   1  19,  142 

crystalline  form  of,  119.   140 
in  urinary  sediments 
Hematuria,  297 

toporphyrin,    [56,    17.?.   174,  282 
iti   urine.  282,  312 
Ila-niin  crystals,   form  of,   164 

test,   163 
Haemochromogen,  156,  173 
Haemoglobin,   151,   156,  170,  282 
carbon  monoxide,   156,   171 
decomposition  of,  156 
diffusion  of,  162 
met,  156,  172 
oxy,  156,  169 
quantitative      determination      of, 

174.    1/8 
reduced,  1 70 
Hemoglobinuria,   297 
Hammerschlag's     method     for     deter- 
mination    of     specific     gravity     of 
blood.  160 
Hammarsten's  reaction,    ijr.   300 

reagent,  preparation   of,    121,   300 
Heintz    method    for    determination    of 

uric  acid,  350 
Heller's  test  for  blood  in  urine,  298 
Heller-Teichmann   reaction   for  blood 

in  urine.  298 
Heller's  ring  test  for  proteid.  47,  290 
Hemi-cedulose.   2 
Herter's  naphthaquinone   reaction   for 

indol  and  skatol.   136.   137 
Heteroproteose.  43 
Heteroxanthin,  238 
Hexoses,   1,   3 

Hippuric  acid.    127,    128,   237,   25s 
crystalline  form  of.  256 
experiments  on,   127,  256 
formula   for,    128,   255 
in  urinary  sediments.   325 
melting-point   of,   257 
separation  of,  from  urine.  256' 
solubility  of.  257 


Hippuric   acid,   sublimation   of,  257 
synthesis  of,  1 27 

llistidin.   65,    79,    107 

hydrochloride,      crystalline      form 
of,    79 

tion   for  tyrosit 
;entisic  acid.   •>,   262 
formula  for,  262 
I [opkins  Cole  reaction,  45 

on    solutions,    45 
on  solids,  51 
Hopkins-Cole  reagent,  preparation  of, 

45 
Hufner*s  urea   apparatus.   354 
Human    fat,   composition   of 
Huppert's   reaction    for  bile   pigments, 

1  -•  1 .   300 
Hurthle's  experiment,  219 
ll>. lime    casts    in    urinary    sediments, 

328,    332 
Hydrobilirubin,  detection  of,  in  feces, 
1 45 
extraction  of,  145 
Hydrochloric  acid   test    for   formalde- 
hyde,  195 
Hydrogen  peroxide  in  urine.  238,  280 

detection  of,  in  milk,   196 
1  [ydrolysis  of  cellulose,  29 
cerebrin,    224 
collagen,  200 
dextrin,  27 
glycogen,    215 
inulin,  26 
saccharose,  20 
starch.    24 
Hyperacidity,   84 
Hypoacidity,  84 
Hypobromite  solution,  preparation  of, 

35i 

Hypoxanthin.  207,  217,  238 

formula  for,  21 1 
Hypoxanthin    silver    nitrate,    crystal- 
line form  of,  216 

Indican.    120,    130,  254 

formula    for,    130.    254 

TafTe's  test  for.  255 

Obermayer's   test   for.   25; 

origin  of,  129,  130 
Indigo-blue,    130,    255 

formula  for,   130,   255 
Indigo  in  urinary  sediments,  318,  327 
Indol.  65,    77-    129,    132,    MO 

formula  for.    129 

origin  of,  120.   140 

tests  for,  136 
Indol-amino-propionic  acid.  77 
Indoxyt,   129 

formula    for.    120 


406 


INDEX. 


lndoxyl,  origin  of,   129,   130 

potassium   sulphate    (see   Indican, 
pp.    129-130,   254.) 
Indoxyl-sulphuric   acid.    129,    237,    253 

formula  for,    129 
Inorganic     physiological     constituents 

of  urine,   238 
Inosinic  acid,   207,   211 
Inosit,    1,   207,  282 

in  urine,  282,  314 
Inulase,  25 
Inulin,   2,   23 

action  of  amylolytic  enzymes  on, 

25 
Fehling's  test  on.  26 
hydrolysis  of,  26 
iodine  test  on,  26 
reducing  power  of,  25 
solubility   of,   25,   26 
sources   of,   25 
Invertin,  20 
Inverting  enzymes,  34 
Iodine   test,   24 
Iodine-sulphuric  acid  test  for  choles- 

terin,    124,    223 
Iodoform  test  for  alcohol,  21 
Iron  in  blood,  156,   161 

detection  of,  161 
in   bone   ash,    204,    205 

detection  of,  204,  205 
Iron  in  proteid,  42 
Iron   in   urine,   238,   280 

detection  of,  280 
Isomaltose,   2,    18 

Jaffe's  reaction   for  creatinin,   253 
Jaffe's  test  for  indican,  255 
v.     Jaksch-Pollak     reaction     for    mel- 
anin, 316 
Jolles'  reaction  for  proteid,  48,  292 

reagent,    preparation    of,    48,    292 
Juice,   gastric,    83-86 

pancreatic,    106-109 

Kephalin,  220,  222 
Kephyr,   19 
Keratin,   43,    62,    197 

experiments   on,    197 

solubility  of,   197 

sources  of,    197 

sulphur  content  of,   197 
Ketone,  1,  7 
Ketose,    1 
Kjeldahl  method  for  determination  of 

nitrogen,   359 
Knop-Hiifner  hypobromite  method  for 

determination  of  urea,   351,   353 
Koumyss,    19 


Kulz's     test     for     /3-oxybutyric     acid, 

309 
Kynurenic   acid,   23-,  262 

formula  for,   262 

isolation  of,  from  urine,  263 

Lactalbumin,    187,    190 

quantitative      determination      of, 
383 
Lactic   acid,    19,   84,   207 

ferric  chloride  test  for,  94 
in   muscular   tissue,   207,   208 
in    stomach    contents,    94,    95 
tests  for,  94 
Uffelmann's  test  for,  94 
Lacto-globulin,    187,    190 
Lactometer,   determination   of   specific 

gravity  of  milk  by,  380 
Lactosazon,       crystalline       form       of, 

Plate  III,  opposite  p.  5 
Lactose,   2,    19,    187,   189,  313 
experiments   on,    19 
fermentation    of,    19 
in  urine,  282,  313 
quantitative  determination  of,  383 
Lffivo-a-prolin,    74 
Lxvulose,   3,    14 

in  urine,  282,  313 
methyl-phenylhydrazin     test     for, 

IS 

phenylhydrazin  test  on,   15 

Seliwanoff's    reaction    for,    15 
Laiose  in  urine,   282,   315 
Laked  blood,  148,  159 
Laky  blood,   161 
Laurie  acid,   187 
Laurin,  98 
Lecithin,    116,    117,    150,   220 

acrolein  test   on,  223 

decomposition    of,    220 

experiments  on,  223 

formula  for,   221 

microscopical  examination  of,  223 

osmic  acid  test  on,  223 

preparation  of,  222 

test  for  phosphorus  in,  223 
Legal's  reaction  for  indol,  137 
Legal's  test  for  acetone,  305 
Legumin,  78 
Leucin,   65,    107.    150 

crystalline   form   of  impure,   326 
pure,  69 

experiments  on,  82 

formula  for,  69 

in  urinary  sediments,  318,  326 

microscopical    examination    of.  82 

separation  of,  from  tyrosin,  81 

solubility    of,    82 

sublimation   of,   82 


INDEX. 


407 


11  ytes,    1  (8,    1  56 

tinting  the,  180,  (84 
number  of,  per  cubic  mm.,   156 
size  of,  156 

variation  in  number  of,  156,  157 
I  ,euco<  vtosis,    1  56 

urn,  2,  37 
Lieben'a  test   for  acetone,  305 
Lieberkuhn's    jelly     (see    Alkali     al- 

buminate,  p.  57  I 
Liebermann-Burchard  test  for  choles- 

terin,   1  -•  t.  aa  1 
Lieberrnann'a  reaction,  46 
I .ipase,  84.  86- 

Lipoids  of  nervous  tissue,  220,  222 
Lipolytic  enzymes,   34 
"Litmus-milk"  tesl    for  steapsin,    115 
Lugol's   solution,    preparation    of,    304 
Lysin,  65,  78,  107 
Lysin  picrate,  crystalline  form  of,  ~^ 

Magnesia     mixture,     preparation     of, 

270 
Magnesium  in  urine,   238 

phosphate    in    urinary    sediments, 

327 
Maltase,   18 
Malto-dextrin,  35 

Maltosazon,  crystalline  form  of,   Plate 

III,  opposite  p.  5 
Maltese,   2.    18 

experiments  on,   18 
Marshall's  urea   apparatus,   352 
Melanin  in  urine,  282,   315 

urinary  sediments,  327 
Melting-point   apparatus.    103 

of  fats,  determination  of,  104 
Messinger-Huppert     method     for     de- 
termination  of  acetone,   374 
Methaemoglobin,   156,   172 
Methyl-mercaptan,    120.    131 
Methyl-pentose   (see  Rhamnose,  p.  2) 
i-methylxanthin,   238 
Micro-organisms      in      urinary      sedi- 
ments,   328.    339 
Milk.    187 

detection    of    calcium    phosphate 
in.   193 
lactose  in,   194 
preservatives   in,    195 
difference    between     human    and 

Cow's,    188 

experiments  on,   190 

formation  of  film  on,   187,  191 

freezing-point   of,    187 

guaiac  test  on,  19.1 

influence   of   rennin   on,    188,    192 

isolation  of  fat  from,   194 


Mill.,      microscopical      appearance     of, 
IMS.     [90 

preparation   of   ca  rom, 

19a 
pi "i"  rtii  1  inogen  of,   190 

quantitative    analysis   of,   380 

iction  of,   187,  190 
separation  of  coagulable  proteids 

of.     I93 

Specific    i;ra\  ity   of,    187,    191 
Millon  11,    44 

reagent,   preparation   of,   44 
Mohr's    method    lor   determination   of 

chlorides,    ;;i 
Molisch's  reaction,   4 
Molybdic  solution,  preparation  of,  37 
Monosaccharides,  1,  2,  3 

classification  of,   1 
Morner-Sjoqvist-Folin  method  for  de 

termination    of    urea.    356 
Morner's    reagent,    preparation   of,   82 

test   for  tyrosin,   82 
Motor  and  functional  activities  of  the 

stomach,  93 
Mucin,  33,  36,  43,  61,  62 
biuret  test  on,  36 
hydrolysis   of,    37 
isolation  of,  from  saliva,  36 
Millon's  reaction  on,  36 
Mucoid,  43,  61,  62,  198 
experiments  on,   199 
hydrolysis  of,   199 
in  urine.  264.  296 
preparation  of,  from  tendon,   199 
Murexid  test,  249 
Muscle  plasma,   206,   212,   213 

formation  of  myosin  clot  in, 

206 
fractional  coagulation  of,  206 
preparation   of   212,   213 
reaction  of.  212 
Muscular  tissue,   206 

commercial    extracts    of,    210 
experiments  on  "  dead,"  214 

"  living,"  212 
extractives   of,   207.   215 
formulas    of    nitrogenous   ex- 
tractives of,   210 
glycogen  in.   207,  208 
lactic   acid   in,    207,   208 
pigment  of,  210 
preparation        of        glycogen 
from,    215 
muscle      plasma      from, 

2\  2.    213 

proteids   of,    206 
reaction  of  living,  208 
separation       of       extractives 
from.   215 


406 


INDEX. 


Myoh<ematin,    210 
Myosin,  206 

biuret   test  on,  214 

coagulation  of,  214 

preparation  of,  214 

solubility  of,  214 
Myosinogen,  206 
Myristic  acid,   187 
Myristin,   98 

Xencki  and  Sieber's  reaction  for  uro- 

rosein,  316 
Nervous  tissue,  220 

constituents  of,  220 
experiments     on     lipoids     of, 

222 
lipoids   of,   220,   222 
percentage   of   water  in,    220 
prosphorized   fats  of,  220 
proteids  of,  220 
Neurokeratin,  220 

Xeutral  olive  oil,  preparation  of,   101 
Neutral  sulphur  compounds,  237,  259 
Nitrates  in  urine,   238,  280 
Xitrites    in    saliva,    test    for,    37 
Nitrogen,  42 

importance  of,   in  sustaining  life, 

42  _ 
in  urine,   quantitative   determina- 
tion of,  359 
Xitrogenous    extractives    of    muscular 
tissue,  207 
formulas  for,   210 
Nitroso-indol  nitrate  test,  137 
Non-nitrogenous    extractives    of   mus- 
cular tissue,  207 
Normal  urine,   226 

characteristics  of,  226 
constituents  of,  237 
experiments  on,  226-281 
Nucleic  acid,  62 
Nucleins,   62.   86,   87,   220 
Xucleo  albumin,  43 
Nucleo  proteid,   43,   61,   62,   220,   237, 
282,  296 
in  bile,  121 
in   feces,    146 
in   nervous  tissue,  220 
in  urine,  9,  237,  264,  282,  296 

tests  for,  297 
occurrence  of,  62 
Ott's    precipitation    test    for, 
297 
Nylander's    reagent,    preparation    of, 
10,  288 
test,  9,   288 

Obermayer's  test  for  indican,  255 
reagent,   preparation    of,    255 


"  Occult  "  blood  in   feces,    142 

tests   for,    144 
Olein,  98 
Olive   oil,    100 

emulsification  of,   101 
neutral,    preparation    of,    101 
Orcin  test,    17 
Organic  acid  radical,  65 
Organic   physiological   constituents   of 

urine,    237 
Organized  urinary  sediments,  328 
Osborne-Folin  method  for  determina- 
tion  of   total  sulphur  in  urine,   363 
Ossein,  204 

preparation  of,  204 
Osseoalbumoid,  43,  204 
Osseomucoid.   43,   62,  204 

chemical   composition   of,    62 
Osseous  tissue,  204 

experiments  on,  204 
Ott's   precipitation   test   for   detection 

of  nucleoproteid   in   urine,   297 
Ovoglobulin,  43 

Oxalated  plasma,  preparation  of,  167 
Oxalic   acid,   237,   258,    378 
formula  for,  258 
in  urine,  237,  258 
quantitative        determination 
of,    378 
Oxaluria,   258 
Oxaluric    acid,    237,    264 
Oxidative   enzymes,    34 
Oxyacids,  129,  135 
tests  for,   138 
/3-oxybutyric   acid,   282,   308 
formula  for,   308 
Kiilz's  test  for,  309 
origin   of,    309 
polariscopic  examination  for, 

309 
quantitative        determination 
of,  375 
Oxyhemoglobin,  156,  169 

Reichert's  method  for  crystalliza- 
tion  of,    167 
crystalline  forms  of,   152-155 
Oxymandelic  acid,  237,   262 
Oxyproteic  acid,  237,  259,  317 

Palmitic  acid,  97,    102,    103.    109 
crystalline  form  of,   102 
experiments    on,    103 
formula  for,  97,  109 
preparation  of,   102 

Palmitin,  98 

Pancreatic   digestion,    106 

general  experiments  on,   ill 
products  of,   107,   no 

Pancreatic  juice,   106-109 


INDEX. 


4O9 


Pancreatic    juice    artificial,    prepara 

tion   of,    no 
daily  excretion  of,   107 
enzymes  of,  107 
freezing  point  of,  107 
mechanism    of   secretion   of, 

106 
reaction  of,   106 
solid  content  of,  107 
specific  gravity  of,  107 
Pancreatic    retrain,    107.    109 
experiments  on,    1 1 5 
resol  sulphuric  acid,  237,  253 

Parallactic    acid.    go8,    238,    265 
Paramyosinogen,  206 
Paraoxyphenylacetic    acid,    129,    136, 
237,  261 

Paranxyphenyl-o-amino-propionic 

acid,   66 
Paraoxyphenylpropiotric      acid,      129, 

i  36,   337,  261 

Paraphenelenediamine     liydrochloride, 

196 
Parasites,    141 
Paraxanthin,  238 
Parietal  glands,  84 
Parotid     glands,      characteristics     of 

saliva   secreted   by,   32 
Pathological  constituents  of  urine,  282 
Pathological  urine,  226,  282 
constituents  of,  282 
experiments  on,  283-317 
Pektoscope,    233 
Pentoses,   2,   16,  311 

experiments   on,    16 
in  urine,  282,   311 
tests  for,  311 
Pepsin,  85,  238 

action  of,  influence  of  bile  upon, 

influence    of    different    acids 
upon,  85,  92 
metallic  salts  upon,  92 
temperature  rpon,  91 
conditions  essential  for  action  of, 

85,    91 
differentiation    of,    from    pepsin- 
ogen, 92 
formation  of,  85 
digestive  properties  of,  85 
most    favorable    acidity    for    ac- 
tion of,  85,  91 
proteolytic  action  of,  85 
Pepsin-hydrochloric    acid,    91-93 
Pepsinogen,   85,  87 

differentiation    of,    from    pepsin, 

02 
formation    of,    85 
extract  of.   preparation   of,  87 


Peptic  proteolysis,  85 

products  of,  85 
relation    of,    to    tryptic    pro- 
ysis,    86 
Peptone,  43,  57,  65,  107,  282, 
ampho,  43,  57 
anti,  43.  58 
differentiation  of,  from  proteoses, 

58 
experiments  on,   58,   60 
in  urine,  29  \ 

tests  for,  295 
separation  of,  from  1  >  r  ■  ■  ■  • 
Pettenkoper's  test  for  bile  acids,   uj, 
301 
Mylius's        modifica- 
tion   Of,     122.    301 

Neukomm's    modifi- 
cation of,  122,  301 
Phenaceturic  acid,  238,  266 
Phenol,  65,   129 

tests  for,   138 
Phenolphtnalein  as  indicator,  89 

preparation   of,   89 
Phenol-sulphuric  acid,  237,  253 
Phenyl-a-amino-propionic   acid,    73 
Phenylalanin,   65,   73 
Phenyldextrosazon,  5 

crystalline  form  of,  Plate  III.  op- 
posite p.   5 
Phenylhydrazin,    5 

acetate  solution,  preparation  of,  5 
mixture,  preparation   of,   5 
reaction,    5 

Cipollina's  modification  of,  6 
Phenyllactasazon.      crystalline       form 

of,   Plate  III.  opposite  p.   5. 
Phenylmaltosazon  crystalline  form  of, 

Plate  III.  opposite  p.  5 
Phosphates   in   urine,    238.    275 
detection  of,   278 
experiments    on.    277 
quantitative        determination 
of,    367 
Phosphocarnic     acid.     207.     211.     238, 

266 
Phospho-proteid,  43,   190 
Phosphorized  compounds  in  urine.  238 
Physiological     constituents    of    urine, 

237 
Pigments  of  urine,  238,  266 
Pine  wood  test  for  indol.    137 
Piria's  test  for  tyrosin.  82 
Polariscope,    use    of,    in    detection    of 
conjugate  glycuronates,  310 
in  determination  of 

13.  340 
/3-oxybutyric  acid,  376 
Polysaccharides,  2.  21 


410 


INDEX. 


Polysaccharides,   classification  of,  2 

properties   of,   21 
Posner's    modification    of    biuret    test, 

46 
Potassium   in  urine,   238 
Potassium  indoxyl-sulphate  (see  Indi- 
can,  pp.    129,   130,  254) 
formula  for,   130,  254 
origin  of,  129,   130 
tests  for,  255 
Primary  proteoses,  58,  59 
Prolin,   65,    74 

crystalline    form    of    laevo-a-,    74 
crystalline    form    of    copper    salt 
of,  75 
Prosecretin,    106 
Protagon,   220,   221 

preparation  of,  222 
Proteids,   42,  282 

acetic  acid  and  potassium  ferro- 

cyanide  test  for,  49 
action  of  alkaloidal  reagents  on, 

47 
action   of  metallic   salts   on,  47 
mineral     acids,     alkalis     and 
organic  acids  on,  47 
Adamkiewicz   reaction   on,   45 
biuret  test  on,   45 
chart  for  use  in  review  of,  63 
chemical    composition    of,    42 
classification  of,  42 
coagulation  or  boiling  test  for,  49 
color  reactions  of,  43 
decomposition   of,   65 
by   hydrolysis,    65 
by  oxidation,  65 
products   of,   65 

experiments  on,  80 
separation  of,   80 
study  of,  65,  80 
derived  simple,  43,  55 
formation   of  fat  from,   99 
formulas  of,  42 
Heller's  ring  test  on,  47 
importance  of,  to  life,  42 
Hopkins-Cole   reaction   on,   45 
in  urine,  282,  289 
tests  for,  290 
Liebermann's   reaction   on,   46 
Millon's  reaction   on,   44 
molecular  weights  of,  42 
native  simple,   42 
Posner's  reaction  on,  46 
precipitation   of,   by  alcohol,    51 
alkaloidal  reagents,  47 
metallic  salts,   47 
mineral  acids,  47 
precipitation  reactions  of,  46 


Proteids,     quantitative     determination 
of,   in   milk,   382 
review  of,  63 

salting-out  experiments   on,  49 
scheme  for  separation  of,  64 
xanthoproteic   reaction   on,  44 
Proteids,  coagulated,  60 

biuret  test  on,  61 
formation  of,  60 
Hopkins-Cole  reaction  on,  61 
Millon's  reaction  on,  61 
solubility  of,   60,   61 
xanthoproteic     reaction     on, 
61 
Proteid-coagulating  enzymes,   34 
Proteids,   compound,  43,   61 
classes  of,  43,  61 
experiments  on,   199 
nomenclature   of,    61 
occurrence   of,   62 
Proteid-cystin,   7y 
Proteids  of  milk,    187,    190 

quantitative        determination 
of,   382 
Proteoids,  43,   62 
Proteolytic  enzymes,   34 
Proteolysis,  peptic,  85 

tryptic,    86,    107 
Proteose,  43,  65,   107,  282,  294 

v.    Aldor's   method   for   detection 

of,  296 
biuret  test  on,  59 
coagulation  test  on,  59 
deutero,  43,   57,  282 
differentiation   of,   from  peptone, 

58 
experiments  on,  59,  295 
hetero,   43,    57,   282 
in  urine,  282,  295 
tests  for,  295 
potassium   ferrocyanide   and  ace- 
tic acid  test  on,  59 
powder,  preparation  of,  59 
precipitation  of,  by  nitric  acid,  59 
by  picric  acid,   59 
by  potassio   mercuric  iodide, 

59 
by   trichloracetic   acid,    59 
primary,  59 
proto,  43,  57 
Schulte's    method    for    detection 

of,  295 
secondary,    59 

separation   of,  from  peptones,  59 
Protoproteose,  43,  57 
Proteoses  and  peptones,  57 
separation  of,  59 
tests  on,  59 
Proteose-peptone,   58 


INDEX. 


411 


ne  peptone,  coagulation  u  si  on, 
58 
experiment!  on,  5s 
Millon'a  reaction  on,  58 
precipitation  of,  by  nitric  acid,  58 
by  pi<  ric  acid,  5K 
Prothrombin,   157,  158 
Pseudo  globulin,  [49,  207 

aines  and  leucomaines  in  urine, 
338,  369 
Ptyalin,  33,  34 

activity  of,  in  stomach,  35 
inhibition  of  acth  ity  of,  35 
nature  of  action  of,  34 
products  of  action  of,  34 
Purdy's  method   for  determination  of 

dextrose,   347 
solution,    preparation    of,    347 
Turin   bases,   62,   238,   269 

in   urine    quantitative    determina- 
tion of,   377 
Pus  casts  in  urinary  sediments,  328,  336 
Pus   cells    in    urinary    sediments,    328, 

329 

Putrefaction,   indican  as  an  index  of, 

129 
Putrefaction    mixture,    preparation    of 

a,  130 
Putrefaction    products,    129 
experiments  on,   130 
most    important,    129 
tests    for,    136 
Pyloric  glands,   83 

Pyrocatechin-sulphuric   acid,   237,   253 
a-pyrrolidin-carboxylic  acid  (see  Pro- 
lin,    pp.    65,    67) 

Qualitative    analysis    of    the    products 
of  salivary  digestion,  41 
stomach    contents,    95 

Quantitative   analysis  of  blood,   386 
of  gastric  juice,  383 
of  milk,  380 
of  urine,   344 

Quantitative     determination     of     am- 
monia in  urine,   357 
acetone  in  urine.  374 
acidity  of  urine,  376 
ash  of  milk,  382 
casein  of  milk,  382 
chlorides   in   urine,    371 
creatinin,    369 
dextrose    in    urine,    345 
fat   in   milk,   380 
lactalbumin   in   milk,   383 
lactose  in  milk,   383 
nitrogen  in  urine,  359 
oxalic  acid  in  urine,  378 
j8-oxybutyric    acid    in    urine, 
3/5 


Quantitative    determination    of    phos 

phoniS    in    urine. 

proteid  in  milk,  382 
proteid   in  urine,  344 
purin   bases   in   urine,   377 

sulphur   in    urine,    .t'.i 

total  solids  in  milk.  381 
total  solids  in   urin< 

1111  .1    111    urine.    33  1 
uric    acid    in    urine, 
Quevcnnc    lactometer,    determination 

of  specific  gravity   of   milk  by,   380 

Raffinose,  2,  21 

Reaction    of    tile    urine,    228 
Reduced    alkali -lnematin,    173 

Reduced   haemoglobin,   170 
Reichet's    method    for    crystallization 
of  oxyhemoglobin,  167 

Remont's     method     for     detection     of 

salicylic    acid   and   salicylates,    195 
Rennin,  84,  86 

action    of,    upon    caseinogen,    86, 
188 

experiments  on,   93,  95 

influence  of,  upon  milk,  93,    192 

in    gastric    juice,    absence    of,    86 

nature  of  action  of,  86 

occurrence  of,  86 
Rennin,  pancreatic,  107,  109 

experiments  on.   115 
Reticulin.  43 
Reynolds-Gunning    test    for    acetone, 

306 
Rhaninose.   2,    17 
Ring  test   for  urobilin.  269 
Robin's  reaction   for  urorosein,  316 
Robert's  ring  test  for  proteid,  48,  291 

reagent,    preparation    of,    48,    291 
Rubner's  test  for  lactose  in  urine,  313 

Saccharide  group,  2 
Saccharose,   2.    19 

experiments  on,  20 

inversion  of,  20 

production  of  alcohol  by  fermen- 
tation of,  20 
Saliva.  32 

alkalinity  of,  33 

amount    of,    33 

bacteria  in,  35 

biuret  test  on.  36 

calcium  in.   33,  37 

chlorides  in,  37 

constituents  of,  33 

digestion  of  dry  starch   by,   38 

digestion  of  inulin  by,    j8 

digestion  of  starch  paste  by,  38 

enzyme  contained  in.   34 


412 


INDEX. 


Saliva,  excretion   of  potassium  iodide 
in,  40 
inorganic  matter  in,  tests  for,  37 
Millon's  reaction  on,  36 
mucin  from,  preparation  of,  36 
nitrites  in,  test  for,  37 
phosphates  in,  test  for,  37 
potassium  sulphocyanide  in,   37 
reaction  of,   36 
secretion  of,   32 
sulphocyanide    in,    tests    for,    37 
specific  gravity  of,   36 
sulphates  in,  test  for,  37 
Salivary  digestion,   32 

influence    of   acids   and    alka- 
lis on,   35,   39 
dilution  on,   39 
metallic   salts   on,   40 
temperature  on,   39 
nature  of  action  of  acids  and 

alkalis    on,    40 
qualitative    analysis    of    pro- 
ducts of,  41 
Salivary  digestion  in  stomach,  35 
Salivary  glands,  32 
Salivary   stimuli,    32 
Salkowski-Autenrieth-Barth       method 
for  determination  of  oxalic  acid  in 
urine,    378 
Salkowski's  method  for  determination 

of  purin  bases,   377 
Salkowski's  test  for  cholesterin,    125 

creatinin,    253 
Salted  plasma,  preparation  of,    167 
Salting-out    experiments    on    proteids, 

49 
Sarcolactic   acid,   208 
Schalfijew's    method    for    preparation 

of  hffimin,  163 
Scheme  for  analysis  of  biliary  calculi, 
124 
bone   ash,   205 
stomach   contents,   95 
urinary   calculi,    343 
separation   of  carbohydrates,   31 
proteids,  64 
Scherer's   coagulation   method   for   de- 
termination   of    albumin    in    urine, 
344 
Schiff's  reaction  for  cholesterin,   125, 
224 
uric    acid,    249 
Schulte's     method     for    detection     of 

proteose  in  urine,  295 
Schweitzer's    reagent,    action    of,    on 
cellulose,  29 
preparation  of,  29 
Secondary  proteoses,  59 
Secretin,    106 


Serin,   65,   74 

crystalline  form  of,  75 
formula  for,  75 
Serum  albumin,  42,  52,   148,   149,  282 
289 
in   urine,  282,  289 
tests   for,  290 
Serum  globulin,  43,  148,  149,  282 
in  urine,  282,  289,  293 
tests  for,  293 
Sherman's  compressed  oxygen  method 
for   determination    of   total   sulphur 
in   urine,  365 
Sherrington's  solution,  preparation  of, 

181 
Silicates  in  urine,  238,  280 
Skatol,  65,   129,   132,   140 

tests   for,    137 
Skatol-carbonic  acid,   135,   136 

test  for,   138 
Skatol-sulphuric   acid,    237,    253 
Skeletins,    63 
Smith's    test    for    bile    pigments,    122, 

301 
Sodium   and  potassium  in   urine,   238, 

279 
Sodium    alizarin    sulphonate    as    indi- 
cator, 90 
preparation  of,  90 
Sodium   hydroxide   and   potassium   ni- 
trate fusion  method  for  determina- 
tion   of    total    sulphur    and    phos- 
phorus  in  urine,    364,    368 
Sodium  hypobromite  solution,  prepar- 
ation of,   351 
Solera's     reaction     for     detection     of 
sulphocyanide  in  saliva,  38 
test  paper,  preparation  of,  38 
Soxhlet    apparatus    for    extraction    of 

fat,  380 
Soxhlet    lactometer,   determination    of 

specific  gravity  of  milk  by,  380 
Spectroscope,  use  of,  in  detection  of 

blood,   169 
Spermatozoa     in     urinary     sediments, 
328,    338 
microscopical    appearance   of   hu- 
man, 338 
Spiegler's    ring    test    for    proteid,    48, 
291 
reagent,  preparation  of,  48,  291 
Standard     ammonium     sulphocyanide 
solution,    preparation    of,    373 
argentic   nitrate   solution,   prepar- 
ation of,  371 
uranium  acetate  solution,  prepar- 
ation of,  367 
Starch,  2,  22 

action  of  alcohol  on  iodide  of,  24 


INDEX. 


41  j 


Starch,   action  of  alkali   on  iodide   of, 
heal   "ii   iodide  of,  24 

dry,  digestion   of,   by   amylopsin, 

1 09,    1 1  1 
dry.  digestion  of,  by  ptyalin,  38 
experiments  on,  -•-■ 

iodine  test    for,   -( 

microscopical  characteristics  of,  22 
microscopical   examination   of,   24 
potato,  preparation  of,  22 
solubility  of,  -4 

various   forms  .of.   23 
Starch  group,  2 
Starch  paste,  action  of  tannic  acid  on, 

25 
diffusibility  of,  25 

digestion     of,    by     amylopsin, 
108,  112 
by    ptyalin.    34.    38 
Fehling's  test  on,  24 
hydrolysis  of,  24 
preparation    of,    24 
Steapsin,   107,    100 

experiments  on.    1  1  5 
ethyl-hutyrate  test  for,   115 
"litmus-milk"  test   for,   115 
Stearic  acid,  221 
Stearin.  98,  187 
Stellar  phosphate.   19.3,  321 
Stokes'   reagent,   action   of.    170 

preparation    of.    170 
Stomach,    motor    and    functional    ac- 
tivities of,  93 
Stomach  contents,  lactic  acid  in,  tests 
for,    94 
qualitative    analysis    of,    95 
Stone-cystin,   77 
Sublingual    glands,    characteristics    of 

saliva   secreted  by,   32 
Submaxilary  glands,  characteristics  of 

saliva  secreted  by,  32 
Sucrose  (see  Saccharose,  p.  19) 
Sulphanilic  acid.  316.  317 
Sulphates  in  saliva,  test  for.  37 
Sulphates  in  urine.  238,  271 
experiments  on.  273 
ethereal.  272.  273 

quantitative     determina- 
tion   of.    363 
inorganic.    272 

quantitative      determina- 
tion of,  362 
total,  quantitative  determina- 
tion  of.   361 
Sulphocyanide    in    saliva,    significance 
of.  33 
ferric    chloride   test    for.    37 
Solera's    reaction    for.    38 


Sulphocyanides  in  urine,  22,7 
Sulphur  in  proteid,  52 

loosely  combined,  test  for,  52 
in    urine,   quantitative   determina- 
tion  of,   361 
aeid.    52 

lead  blackening,  ?-• 
mercaptan,  52 
neutral,    52 
oxidized,  52 
unoxidized,   52 

Tallow,    bayberry,     saponification     of, 

102 
Tallquist'a    haemoglobin    scale. 

mination  of  haemoglobin  by,  180 
Tannic  acid,  influence  of,  on  dextrin, 
28 
on  starch,  25 
Tanret's  reagent,   preparation   of,   48, 

293 
Tanret's  test,  48,  293 
Tartar,  formation  of,  33 
Taurin,    117,    207.   211 

formula   for,    117.    211 
preparation   of,    125 
Taurin    derivatives,    237 
Taurocholic  acid,   117 

group,  117 
Teichmann's    crystals,    form    of    Csee 

hsmin  crystals,  p.   164) 
Tendomucoid.  43.  61.  62,  199 
biuret  test  on,  199 
chemical  composition  of,  62 
hydrolysis  of,   199 
loosely  combined  sulphur  in.  test 

for.   199 
preparation  of,  199 
solubility   of.    199 
Thoma-Zeiss  hxmocytometer,  180 
Thrombin.    157,    158 
Tincture  of  iodine,  preparation  of,  394 
Tissue,   adipose,   experiments  on,    100, 
205 
connective,    197 

white  fibrous,   197 

composition    of.    198 
experiments  on.  198 
yellow   elastic.    201 

composition    of,    201 
experiments  on,  201 
epithelial.   197 

experiments  on.    tor 
muscular.  206 

experiments  on,  212 
nervous.   220 

experiments  on,   222 
osseous.  204 

experiments   on,   204 


414 


INDEX. 


Tissue    debris    in    urinary    sediments, 

328,  339 
Toison's  solution,  preparation  of,   181 
Tollens'    reaction    on    conjugate    gly- 
curonates,   310 
galactose,   15 
arabinose,   16 
Topfer's      method      for      quantitative 

analysis  of  gastric  juice,  383 
Topfer's  reagent,  as  indicator,  88 

preparation  of,  88 
Total   solids  of  milk,  quantitative  de- 
termination  of,    381 
of   urine,    quantitative   deter- 
mination  of,   379 
Total    sulphur    of    urine,    quantitative 
determination  of,  363,  364,  365 
phosphorus  of  urine,  quantitative 
determination  of,  368 
Tri-butyrin.   187 
Tri-olein.    98,    187 
Tri-palmitin,  98,  187 
Tri-stearin,  98,   187 
Trichloracetic    acid,    precipitation    of 

proteid  by,   47 
Trioses.    1 
Triple  phosphate,  278 

crystalline  form  of,  278 
formation  of,  277 
Trisaccharides,  2,  21 
Trommer's  test,  8,  285 
Tropreolin  00,  as  indicator,  89 

preparation  of,  89 
Trypsin,    107 

action    of,   upon   proteids,    107 

experiments  on,   111 

influence    of    alkalis    and    mineral 

acids  upon,  107 
nature  of,    107 
pure,  preparation  of,  107 
Trypsinogen,    107,    108 
activation    of,    108 
Tryptic  digestion,  107 

influence   of   bile   on,    112 
metallic   salts  on,    112 
most   favorable  reaction  for, 
in 
temperature  for,    in 
products  of,   107,   no 
Tryptic   proteolysis,  86,    107 
Tryptophan,    45,    65,    77,    107,    in 
bromine   water   test   for,    no 
formula  for,  77 

group  in  the  proteid  molecule,  45 
Hopkins-Cole  reaction  for,  45 
occurrence    of,    as    a    decomposi- 
tion product  of  proteid,  65,  77 
occurrence  of,  as  an  end-product 


of     pancreatic     digestion,     107, 

1 1 1 
Tyrosin,  65,  66,   107 

crystalline   form   of,   68 
experiments  on,  81 
formula  for,  66 
Hoffmann's  reaction  for,  82 
in  urinary  sediments,   319,   326 
microscopical   examination  of,  81 
Morner's  test  for,  82 
occurrence  of,  67 
Piria's   test   for,   82 
salts  of,  68 

separation  of,  from  leucin,  68,  81 
solubility   of,   82 
sublimation  of,  82 
Tyrosin-sulphuric  acid,   82 

v.  Udransky's  test  for  bile  acids,   123, 

302 
Uffelmann's   reagent,  preparation  of,  94 
Uffelmann's    reaction    for    lactic    acid, 

94 
Unknown  substances  in  urine,  282,  316 
Uranium    acetate    method    for    deter- 
mination    of     total    phosphates     in 
urine,   367 
Urate,     ammonium,    crystalline    form 
of,    Plate    VI,    opposite    p.    324 
sodium,    crystalline   form  of,    324 
Urates  in  urinary  sediments,  319,  323 
Urea,  237,  239 

crystalline   form   of,  239 
decomposition      of,      by      sodium 

hypobromite,   241 
excretion   of,   240 
experiments   on,-  242 
formation    of,    240 
formula  for,  239 
furfurol  test  for,  245 
isolation  of,   from   the  urine,   242 
melting-point  of,  243 
quantitative      determination      of, 

35i 
Urea  nitrate,  241 

crystalline  form  of,  242 

formula   for,   241 
oxalate,    241 

crystalline   form   of,   244 

formula    for,    241 
Urethral    filaments    in    urinary    sedi- 
ments,   328,    338 
Uric  acid,  9,  207,  2^7,  245 

crystalline  form  of  pure,  249 

endogenous,   246 

exogenous,   246 

experiments    on,    248 

formula   for,  245 

in  leukaemia,  248 


IXDK.X. 


415 


Uric  acid  in  urinary  sediment-. 

crystalline  Eorin  of, 
Plate  V,  opposite  p. 
347,  333 

isolation   of,    from    the  urine, 
248 

murexid  tesl   for,  349 

origin  "' 

quantitative       determination 

of,    349 
Folin  Schaffer 
method  for,  349 

llrintz    method    for, 

350 
reducing  power  of,  0,  249 
Schiff's  reaction  for,  249 
Urinary  calculi.   3  p> 

calcium  carbonate  in,  341 

oxalate   in.   341 
cholesterin  in,   342 
comnound,    340 
cvstin   in,   34-' 
fibrin    in.    343 
indigo  in,  342 
phosphates  in,  341 
scheme    for    chemical     anal- 
ysis of.   343 
simple,   340 

uric  acid  and  urates  in.  341 
urostealiths  in,  342 
xanthin    in,    342 
Urinary     concrements      (see     urinary 

calculi,  p.    34°^ 
Urinary      concretions      (see      urinary 

calculi,   p.   34°^ 
Urination,   frequency  of,  228 
Urinary  sediments.   318 

ammonium   magnesium   phos- 
phate in.   319 
animal   parasites   in.   339 
calcium    carbonate    in,    321 
oxalate  in,  320 
phosphate  in,  321 
sulphate  in.   322 
casts  in.   332 
cholesterin   in,   325 
collection   of.    318 
cylindroids  in,   337 
cvstin    in.    324 
epithelial  cells  in,   328 
erythrocytes  in.   337 
fibrin    in.     ;  ") 
forei.en    substances   in.    339 
hematoidin   and  bilirubin   in, 

326 
hipnuric   acid   in,   325 
indisro  in.  327 
leucin   and   tvrosin   in.   326 
magnesium  phosphate  in,  327 


Urinary  sediments,  melanin  in, 
micro-organisms  in.  339 
organized,  338 

PUS    cells    in. 

-p.  1  matozoa  in,  338 

tissue    debris    in 

unorganized,  319 

urates    in.    3^3 

urethral    filaments    in.    338 

uric    acid    in.    322 

xanthin  in,  327 
Urine,  326-379 

acetone  in,  302 

acidity  of,  228 

acid  fermentation  of,  230 

albumin  in,  289 

alkaline    fermentation    of,    230 

allantoin  in,  259 

ammonia   in,   270 

aromatic   oxyacids   in,   261 

benzoic  acid  in,  263 

bile   in,   300 

blood  in,  297 

calcium  in,  279 

carbonates  in,   280 

chlorides  in,   274 

collection   of.   235 

conjugate  glycuronates  in,   310 

color  of.  226 

creatinin  in,   250 

dextrose   in,   282 

diacetic   acid   in,   306 

electrical  conductivity  of,  235 

enzymes  in,  265 

ethereal  sulphuric  acid  in,  253 

fat  in,   312 

fluorides  in  280 

freezing-point  of,  232 

general  characteristics  of.   226 

globulin    in,    293 

Haser's   coefficient   for   solids   in, 

379 

hxmatoporphyrin  in,  312 

hippuric  acid  in.   255 

hydrogen   peroxide  in,   280 

inorganic      physiological      consti- 
tuents of,  238 

inosit   in.    314 

iron  in.  280 

lactose  in.  313 

lrcvulose  in,  313 

laiose  in,  315 

leucomaines  in,  269 

T. one's    coefficient    for    solids    in, 

379 
magnesium  in.  279 
melanin    in.    315 
neutral     sulphur     compounds     in, 

259 


416 


INDEX. 


Urine,  nitrates  in,  280 

nucleoproteid  in,  264,  296 

odor   of,   228 

organic  physiological  constituents 
of,  237 

oxalic  acid  in,  258 

oxaluric  acid  in,   264 

/3-oxybutyric  acid  in,  308 

pathological    constituents    of,    282 

paralactic  acid  in,  265 

pentoses   in,   311 

peptone  in,   294 

phenaceturic   acid   in,  266 

phosphates  in,   275 

phosphorized    compounds   in,    266 

physiological  constituents  of,  237 

pigments    of,   266 

potassium    in,    279 

proteids   in,    289 

proteoses  in,  294 

ptomaines  in,  269 

purin  bases  in,  269 

quantitative   analysis  of,    344 

reaction   of,    228 

silicates   in,    280 

sodium   in,   279 

solids  of,  232,  379 

specific  gravity  of,  230 

sulphates  in,  271 

transparency  of,   228 

unknown    substances    in,    316 

urea  in,  239 

uric   acid   in,    245 

urorosein   in,    316 

volatile  fatty  acids  in,  265 

volume  of,  226 
Urobilin,  i.;8,  266,   267 

tests  for,   267 
Urochrome,  238,  266 
Uroerythrin,    238,    266,   269 
Uroferric  acid,  237,  259,  317 
Uroleucic  acid,  237.   262 
Urorosein.  282,   316 

tests  for,  316 


Vegetable     gums,     2 

Veith  lactometer,  determination  of 
specific  gravity  of  milk  by,  380 

Volatile  fatty  acids,   132,   133,   195 

Volhard-Arnold  method  for  determin- 
ation  of  chlorides,    372 

Volume  of  the  urine,  226 

Waxy  casts  in  urinary  sediments,  336 
Weber's    guaiac    test     for    blood     in 

feces,    145 
Weyl's  test  for  creatinin,  252 
White  fibrous  connective  tissue,  197 

experiments   on,    198 
Wirsing's   test    for    urobilin,    268 

Xanthin,  207,  238 

crystalline  form   of,   210 

formula  for,   211 

in   urinary   sediments,    327 

isolation   of,    from   meat   extract, 
217 

Weidel's  reaction  for,  218 
Xanthin  bases    (see   Purin  bases,   pp. 

62,  238,  269) 
Xanthin  silver  nitrate,  217 

crystalline   form   of,   218 
Xanthoproteic   reaction,    44 
Xylose,  2,  17 

orcin  reaction  on,  17 

phenylhydrazin  reaction  on,   17 

Tollens'   reaction   on,    17 

Yellow  elastic  connective  tissue,  201 
composition  of,  201 
experiments  on,  201 

Zappert  slide,   184 

Zeller's  test  for  melanin,  315 

Zeynek  and  Nencki's  hsemin  test,   163 

299 
Zikel  pektoscope,   233 
Zymogen,  85,  107 


Practical  physiological  chemistry 


